Microsystem, microopening film, and system and method for analizing interaction between biomolecules

ABSTRACT

A micro system capable of setting an appropriate amount of stimulation being applied in order to control liquid flow in a channel. The micro system comprises micro-heaters ( 5   b,    5   c ) for applying stimulation to liquid flowing through liquid channels ( 2   b,    2   c ) formed in a plate ( 1 ) and controlling liquid flow by the stimulation from the micro-heaters ( 5   b,    5   c ), and a means for electrically controlling the amount of stimulation being applied to the liquid from the micro-heaters ( 5   b,    5   c ). An appropriate amount of stimulation can be set by electrically controlling the amount of stimulation being applied to the liquid from the micro-heaters ( 5   b,    5   c ) through the control means.

TECHNICAL FIELD

The invention relates to a micro-system for controlling liquid flowthrough a micro liquid channel, a nano-aperture film for detecting andquantifying biomolecular interaction at the level of a single molecule,and a device and method for analyzing biomolecular interaction.

BACKGROUND ART

The progress of nano-technology in recent years has promoted thedevelopment of micro-systems for analyzing samples, or producing areaction, by forming a liquid channel of micron order on chips such asglass, and passing a sample through this liquid channel. Themicro-system has advantages such as the capability of analysis withsmall amounts of samples, which is attracting much attention.

However, in this micro-system, there are problems in that it isdifficult to prepare a valve in a liquid channel for controlling theflow of the sample, and to control the flow of the sample.

In order to solve the problem in the conventional technique, JapanesePatent Publication No. 2002-163022 discloses a method for controllingthe flow, comprising the steps of adding a sol-gel transitionalsubstance transferred by the stimulation of heat such as from anexternal laser etc. to a liquid flowing in the minute liquid channel ofthe micro-system, and applying stimulation to a desired point on theminute liquid channel so as to transform the liquid into a gel.According to this method, without using a complicated valve structure,the liquid flow can be stopped, and the flow amount or the flow rate canbe simply adjusted. When a branch is formed in a part of the channelsand stimulation is applied to a liquid in a channel chosen from thebranched channel, it is possible to choose a direction through whichliquid flows by blocking the channel by turning the substance into agel. By stopping stimulation, the substance turns into a sol and thechannel is opened wide again.

According to the above-mentioned method, however, in the case where theappropriate amount of stimulation is not applied to the liquid, there isa problem in that it is not appropriately turned into the gel, and doesnot block the channel because the amount of stimulation is too little.There is also a problem in that the liquid is heated beyond necessityand it takes time for gelation, when the amount of stimulation is toomuch.

A first object of the present invention is to solve the above-mentionedproblem, and to provide a micro-system which can apply an appropriateamount of stimulation in order to control a liquid flow in a channel.

Moreover, the above-mentioned method comprises: forming a liquid channelon a substrate in advance using conventional ultra-fine processingtechnology; and flowing a liquid through the formed liquid channel. Asto the method of forming this liquid channel, there is, for example, amethod of etching a substrate by a chemical reaction, and of cutting thesubstrate using a photocurable resin or a thermosetting resin. There isa problem in that this method needs complicated process for forming theliquid channel, and it takes time for this method. Moreover, in thismethod it is possible to stop the liquid flowing through the liquidchannel and to adjust the flow amount by gelation, but only the existingliquid channel can be used and the liquid channel itself can not benewly formed or removed. Therefore it has a problem in that it isnecessary to prepare the liquid channel with a different channel formfor each purpose.

A second object of the present invention is to provide a matrix typevariable liquid channel capable of forming and removing a liquid channelin arbitrary positions by freely forming a wall and a valve structure,and a system capable of controlling it. Also to provide a matrix typevariable liquid channel which does not need a liquid channel to beformed on a plate in advance, and a system capable of controlling it.

On the other hand, as a conventional method for detecting andquantifying biomolecular interaction at the one molecule level, thereare mainly the following two methods.

One is a method as shown in FIG. 35 for imaging a fluorescentbiomolecule 306 using an evanescent field 305 generated at an interface303 of a solution 302 by applying a laser light 304 to an interface 303between a glass 301 and a solution 302 to induce total internalreflection. Since the evanescent field 305 is attenuated by the 150 nmpenetration length from the interface 303, partial excitation ispossible. When one fluorescent biomolecule 306 is fixed to the glass 301and the other biomolecule labeled with a fluorescence molecule ofanother fluorescence wavelength is added to the solution 302, it ispossible to take an image of an association and a dissociation of thesebiomolecules (interaction between molecules) by a high sensitivitycamera 307. This single fluorescent molecule imaging method forbiomolecules was developed by the inventors in 1995 (Funatsu, T. et al.(1995), Nature 374, 555-559), and has achieved various success. However,there are the following problems. 1. Unless concentration of thefluorescent biomolecule 306 in the solution 302 is less than 50 nM, itis difficult to observe one molecule. 2. Because fluorescentbiomolecules nonspecifically bind to the glass 301, it is difficult todetect the interaction between molecules. Therefore, detectingbiomolecular interaction at the one molecule level is limited.

The reason for these problems is as follows. The evanescent fieldinduced by total internal reflection generates partial excitation 150 nmin the direction perpendicular to the interface 303, but it is notlocalized in the parallel direction. Therefore each fluorescentbiomolecule 306 can not be imaged in the range of the resolution (about250 nm) of the optical microscope connected to the high sensitivitycamera 307 if the concentration of the fluorescent biomolecule 306 inthe solution 302 is not set to less than 50 nM. Moreover, since thefluorescence 308 is affected by diffraction during transmission to thehigh sensitivity camera 307, the fluorescence 308 emitted by thefluorescent biomolecule 306 expands to a size which is equivalent to thediameter of about 250 nm in the surface of the sample. Therefore, inorder to identify each fluorescent biomolecule 306, the interval of thefluorescent biomolecule 306 fixed to the glass 301 must be more thanabout 250 nm. Therefore, the rate of the area occupied by thefluorescent biomolecule 306 fixed on the glass 301 becomes as small as0.1% or less, which results in a problem of nonspecific adsorption tothe glass 301 of the biomolecule added into the solution 302. Theseproblems are expected to be solved by making smaller domains where thefluorescent biomolecule 306 is excited, and arranging them at a positionaway from the resolution of the optical microscope connected to the highsensitivity camera 307.

The second method of detecting biomolecular interaction at the singlemolecule level is fluorescence correlation spectroscopy (FCS;Fluorescence Correlation Spectroscopy). As shown in FIG. 36, this is themethod of obtaining the fluorescence intensity and diffusion constant ofeach fluorescent biomolecule 313, by narrowing down the laser beam 311to the diffraction limit with a large numerical aperture object lens312, and measuring the fluctuation of the fluorescence intensity of thefluorescent biomolecule 313 which passes through it (Eigen M. andRigler, R. (1994) Proc. Natl. Acad. Sci. USA 91, 5740-5747, publishedJapanese translation of PCT International Publication No. H11-502608).In order to detect only the fluorescence 315 at the focus 314 of thelaser beam 311, a confocal optical system with a pinhole on the imagefocus location of the laser beam 311 is used. If two kinds offluorescence are correlated with each other, it is also possible toanalyze two kinds of interaction between the fluorescent biomolecules313. This method is called fluorescence Cross-correlation Spectroscopy(FCCS) (Rigker, R., Z. et al. (1998), Fluorescence cross-correlation-anew concept for polymerase chain reaction. J. Biotechnol. 63: 97-109).Also in this method, since the irradiation domain spreads to the extentof the diffraction limit of light, the concentration of the fluorescentbiomolecule 313 can be raised only to about 100 nM. In order to observethe biomolecular interaction at higher concentration, partial excitationexceeding the diffraction limit of light is desired.

A third object of the present invention is to solve the above-mentionedproblem, to realize making the irradiation domain of the excitationlight smaller than the wavelength of the excitation light, and toprovide a nano-aperture film to sensitively detect and determinebiomolecular interaction at the level of a single molecule, and a deviceand method for analyzing biomolecular interaction.

SUMMARY OF INVENTION

In a micro-system according to a first aspect of the invention, in orderto achieve the first object, the present invention provides themicro-system comprising a stimulation applying means for applyingstimulation to a liquid flowing in a liquid channel formed in a plate,the liquid flow being controlled by the stimulation from the stimulationapplying means, wherein the stimulation applying means comprises acontrol means for electrically controlling an amount of stimulationapplied to the liquid.

Thus, an appropriate amount of stimulation can be set by electricallycontrolling the amount of stimulation being applied to the liquid fromthe stimulation applying means by the control means.

According to a second aspect of the invention, the present inventionprovides the micro-system, further comprising a stimulation detectingmeans for detecting the amount of stimulation, wherein the stimulationapplying means is a heat source or a light source, and said stimulationapplying means is controlled by said control means based on a signalfrom said stimulation detecting means, in the above-mentioned firstaspect of the invention.

Thus, an appropriate amount of stimulation can be set.

According to a third aspect of the invention, the present inventionprovides the micro-system, wherein the heat source is a micro-heater, inthe above-mentioned second aspect of the invention.

Thus, the stimulation can be certainly applied to the liquid.

According to a fourth aspect of the invention, the present inventionprovides the micro-system, wherein said stimulation detecting means is aheat sensor provided on the liquid channel, in the above-mentionedsecond aspect of the invention.

Thus, the amount of stimulation being applied to the liquid from thestimulation applying means can be certainly detected.

According to a fifth aspect of the invention, the present inventionprovides the micro-system, wherein the heat sensor is a thermo-couple,in the above-mentioned fourth aspect of the invention.

Thus, the heat sensor can be constituted easily.

According to a sixth aspect of the invention, the present inventionprovides the micro-system, wherein the heat sensor is a heat sensitivesemiconductor or an infrared ray sensitive sensor, in theabove-mentioned fourth aspect of the invention.

Thus, the amount of stimulation is certainly detectable.

According to a seventh aspect of the invention, the present inventionprovides the micro-system, wherein the light source is at least onelight emitting element installed in the plate, in the above-mentionedsecond aspect of the invention.

Thus, the light source can be constituted easily.

According to an eighth aspect of the invention, the present inventionprovides the micro-system, wherein the light emitting element isembedded in the plate, in the above-mentioned seventh aspect of theinvention.

Thus, the light emitting element can be arranged near the liquidchannel, and the stimulation can be certainly applied to the liquid.

According to a ninth aspect of the invention, the present inventionprovides the micro-system, wherein the light emitting element isarranged outside the plate, in the plate in the above-mentioned seventhaspect of the invention.

Thus, even if the plate is used once and then thrown away, the lightemitting element can be used repeatedly.

According to a tenth aspect of the invention, the present inventionprovides the micro-system, further comprising an optical guiding pathfor guiding a light from the light emitting element, the optical guidingpath being formed horizontally with a surface of the plate in which theliquid channel is formed, in the above-mentioned ninth aspect of theinvention.

Thus, the light from a light emitting element can be efficientlyintroduced to the liquid channel.

According to an eleventh aspect of the invention, the present inventionprovides the micro-system, further comprising a plurality of lightemitting elements, in the above-mentioned seventh aspect of theinvention.

Thus, the stimulation can be applied in two or more parts where theliquid channels differ.

According to a twelfth aspect of the invention, the present inventionprovides the micro-system, further comprising:

-   -   an energy imparting means for imparting energy to the liquid;        and    -   a change detecting means for detecting a change in a substance        which causes a change by energy from said energy imparting        means, wherein said stimulation applying means is controlled by        said control means based on a signal from said change detecting        means, in the above-mentioned first aspect of the invention.

Thus, only the liquid containing the substance can be easily divided bycontrolling the liquid flow based on a change in a substance whichcauses a change by energy from said energy imparting means.

According to a thirteenth aspect of the invention, the present inventionprovides the micro-system, further comprising an energy guiding path forguiding the energy from the energy imparting means, the energy guidingpath being formed horizontally with a surface of the plate, in theabove-mentioned twelfth aspect of the invention.

Thus, the energy from the energy imparting means can be efficientlyguided to the liquid channel.

According to a fourteenth aspect of the invention, the present inventionprovides the micro-system, wherein the change detecting means is afluorescence detecting element or a light receiving element, in theabove-mentioned twelfth aspect of the invention.

Thus, a change in the substance which causes change by the energy fromsaid energy imparting means is detectable.

According to a fifteenth aspect of the invention, the present inventionprovides the micro-system, wherein the fluorescence detecting element orthe light receiving element is arranged horizontally with the surface ofthe plate, in the above-mentioned fourteenth aspect of the invention.

Thus, a change in the substance which causes change by the energy fromsaid energy imparting means is detectable from a side of the liquidchannel.

According to a sixteenth aspect of the invention, the present inventionprovides the micro-system, wherein the fluorescence detecting element orthe light receiving element is arranged above the liquid channel, in theabove-mentioned fourteenth aspect of the invention.

Thus, change of the substance which produces change by the energy froman energy grant means is detectable from above the liquid channel.

According to a seventeenth aspect of the invention, the presentinvention provides the micro-system, further comprising:

-   -   a stand for mounting the plate; and    -   a positioning means for deciding a position of the plate on the        stand, in the above-mentioned first aspect of the invention.

Thus, the plate can be placed easily in the correct position of thestand by the positioning means. In particular, when the plate is usedonce and then thrown away, the effort of positioning can be saved at thetime of placing the plate correctly.

In a matrix type variable liquid channel according to an eighteenthaspect of the invention, as a means for achieving the second object, thepresent invention provides a matrix type variable liquid channel,comprising two or more stimulation sensitive members arranged on a platein a pattern of a two dimensional matrix.

Thus, since a wall or valve structure can be formed reversibly through asol-gel transition at any position by stimulating the stimulationsensitive members arranged in a pattern of the two dimensional matrix,liquid channels can be easily made. Moreover, since the stimulationsensitive member is stimulated, the gelation rate of a substance havingsol-gel transition properties increases. Furthermore, since the channelshape can be changed freely, preparation of liquid channels havingdifferent channel shapes is not necessary. As the stimulation sensitivemember, the metal pieces which generate heat by stimulation can be used.For example, titanium, chromium, or the like can be used as these metalpieces. Additionally, when a biological reaction is taken into account,it is desirable to use titanium that does not react with a living body.

According to a nineteenth aspect of the invention, the present inventionprovides the matrix type variable liquid channel, wherein saidstimulation sensitive members on the plate are arranged at certainintervals, in the above-mentioned eighteenth aspect of the invention.

Thus, since there is an interval between the stimulation sensitivemembers, gelation at any positions is facilitated.

According to a twentieth aspect of the invention, the present inventionprovides the matrix type variable liquid channel, wherein a size of eachstimulation sensitive member ranges from 2 μm or more to 20 μm or less,in the above-mentioned nineteenth aspect of the invention.

Thus, stimulation sensitive members are stimulated so that a substancehaving sol-gel transition properties can be gelated in response to thesize of the stimulation sensitive members. Additionally, the preferredsize of each stimulation sensitive member ranges from 2 μm or more to 20μm or less. This is because if the size of each is less than 2 μm, thethickness of the wall or valve structure becomes thin by gelation whichis not desirable, while if greater than 20 μm, the thickness of the wallor valve structure becomes thick by gelation, and unless a particularlythick wall or valve structure is required, this is not necessary.

According to a twenty-first aspect of the invention, the presentinvention provides the matrix type variable liquid channel, wherein saidstimulation sensitive members are arranged at intervals from 2 μm ormore to 20 μm or less, in the above-mentioned nineteenth aspect of theinvention.

According to this construction, since an area where a substance havingsol-gel transition properties gelates by stimulation is larger than saidstimulation sensitive members, and a gelling area connects even if saidstimulation sensitive members are arranged at suitable intervals, thewall or valve structure can be formed. Moreover, the preferred intervalbetween said stimulation sensitive members is from 2 μm or more to 20 μmor less. This is because the interval is narrow if the interval is lessthan 2 μm, while it is difficult to connect a gelling area with theinterval over 20 μm.

According to a twenty-second aspect of the invention, the presentinvention provides the matrix type variable liquid channel, wherein saidstimulation sensitive members are formed by vapor deposition,sputtering, Chemical Vapor Deposition (CVD), plating, plasmapolymerization, or screen-printing, in the above-mentioned nineteenthaspect of the invention.

Thus, said stimulation sensitive members can be easily formed on a plateby using vapor deposition, sputtering, Chemical Vapor Deposition (CVD),plating, plasma polymerization, or screen-printing.

According to a twenty-third aspect of the invention, the presentinvention provides the matrix type variable liquid channel, wherein saidstimulation sensitive member is stimulated by applying a voltage orirradiating a light thereto, in the above-mentioned eighteenth aspect ofthe invention.

Thus, since said stimulation sensitive member is stimulated by applyinga voltage or irradiating a light thereto, the temperature of saidstimulation sensitive member can be adjusted and a sol-gel transitioncan be easily initiated.

In a matrix type variable liquid channel system according to atwenty-fourth aspect of the invention, the present invention providesthe matrix type variable liquid channel system comprising:

-   -   a matrix type variable liquid channel which comprises two or        more stimulation sensitive members arranged on a plate in a        pattern of a two dimensional matrix;    -   a detecting means for detecting a substance on the plate;    -   a stimulation applying means for applying stimulation to the        stimulation sensitive members; and    -   a control means for controlling the stimulation applying means        based on the signal from the detecting means.

Thus, said stimulation sensitive members arranged in a pattern of a twodimensional matrix is stimulated by the stimulation applying means sothat a wall or valve structure through a sol-gel transition can beformed reversibly at positions corresponding to said stimulationsensitive members. Therefore, the liquid channel can be easily made.Moreover, since the stimulation sensitive member is stimulated, thegelation rate of a substance having sol-gel transition propertiesincreases. Additionally, since the channel shape can be changed freelyby controlling the stimulation applying means, preparation of a liquidchannel having different channel shapes is not necessary. Furthermore, asubstance can be detected at any positions on a plate, and a desiredsample substance is easily separated or analyzed. As a stimulationsensitive member, metal pieces which generate heat by stimulation can beused. For example, titanium, chromium, or the like can be used as thesemetal pieces. Additionally, when a biological reaction is taken intoaccount, it is desirable to use titanium which does not react with aliving body.

According to a twenty-fifth aspect of the invention, the presentinvention provides the matrix type variable liquid channel system,wherein said stimulation sensitive members on the plate are arranged atcertain intervals, in the above-mentioned twenty-fourth aspect of theinvention.

Thus, since there is an interval between stimulation sensitive members,gelation at any positions is facilitated. Moreover, since the channelshape can be easily changed, a substance can be detected at anypositions on a plate, and a desired sample substance can be easilyseparated or analyzed.

According to a twenty-sixth aspect of the invention, the presentinvention provides the matrix type variable liquid channel system,wherein a size of each stimulation sensitive member ranges from 2 μm ormore to 20 μm or less, in the above-mentioned twenty-fifth aspect of theinvention.

Thus, stimulation sensitive members are stimulated so that a substancehaving sol-gel transition properties can be gelated in response to thesize of the stimulation sensitive members. Additionally, the preferredsize of each stimulation sensitive member ranges from 2 μm or more to 20μm or less. This is because if the size of each is less than 2 μm, thethickness of the wall or valve structure becomes thin by gelation whichis not desirable, while if greater than 20 μm, the thickness of the wallor valve structure becomes thick by gelation, and unless a particularlythick wall or valve structure is required, this is not necessary.Moreover, since the channel shape can be easily changed, a substance canbe detected at any positions on a plate, and a desired sample substancecan be easily separated or analyzed.

According to a twenty-seventh aspect of the invention, the presentinvention provides the matrix type variable liquid channel system,wherein said stimulation sensitive members are arranged at intervalsfrom 2 μm or more to 20 μm or less, in the above-mentioned twenty-fifthaspect of the invention.

According to this construction, since an area where a substance havingsol-gel transition properties gelates by stimulation is larger than saidstimulation sensitive members, and a gelling area connects even if saidstimulation sensitive members are arranged at suitable intervals, thewall or valve structure can be formed. Moreover, the preferred intervalbetween said stimulation sensitive members is from 2 μm or more to 20 μmor less. This is because the interval is narrow if the interval lessthan 2 μm, while it is difficult to connect a gelling area with theinterval over 20 μm. Moreover, since the channel shape can be easilychanged, a substance can be detected at any positions on a plate, and adesired sample substance can be easily separated or analyzed.

According to a twenty-eighth aspect of the invention, the presentinvention provides the matrix type variable liquid channel system,wherein said stimulation sensitive members are formed by vapordeposition, sputtering, Chemical Vapor Deposition (CVD), plating, plasmapolymerization, or screen-printing, in the above-mentioned twenty-fifthaspect of the invention.

Thus, said stimulation sensitive members can be easily formed on a plateby using vapor deposition, sputtering, Chemical Vapor Deposition (CVD),plating, plasma polymerization, or screen-printing. Furthermore, byconstructing the invention in this manner, a system is able to becheaply manufactured.

According to a twenty-ninth aspect of the invention, the presentinvention provides the matrix type variable liquid channel system,wherein said stimulation sensitive member is stimulated by saidstimulation applying means applying stimulation thereto, saidstimulation being the application of voltage, or irradiation of light,in the above-mentioned twenty-fourth aspect of the invention.

According to this construction, since it is configured so thatstimulation may be applied to said stimulation sensitive member byapplying voltage or irradiating a light as said stimulation applyingmeans, the temperature of said stimulation sensitive member can beadjusted and a sol-gel transition can be easily initiated. Furthermore,by constructing the invention in this manner, since the channel shapecan be easily changed, a substance can be detected at any positions on aplate, and a desired sample substance can be easily separated oranalyzed.

In a nano-aperture film according to a thirtieth aspect of theinvention, as a means for achieving the third object, the presentinvention provides the nano-aperture film, comprising a thin film whichdoes not transmit light and in which at least one nano-aperture isformed.

According to this construction, by forming the maximum opening width ofa nano-aperture smaller than the wavelength of the excitation light andirradiating the nano-aperture with excitation light, an evanescent fieldis generated through the nano-aperture. Thus using the evanescent field,it is possible to irradiate a fluorescent biomolecule with an excitationlight in a smaller region than the wavelength of the excitation light.

According to a thirty-first aspect of the invention, the presentinvention provides the nano-aperture film, wherein the thin film iscombined with a transparent plate, in the above-mentioned thirtiethaspect of the invention.

Thus, the manufacturing and handling of a thin film can be improved bysupporting the thin film on the plate. Moreover, since the plate istransparent, it does not prevent the transmission of excitation light.

According to a thirty-second aspect of the invention, the presentinvention provides the nano-aperture film, wherein a plurality ofnano-apertures are provided and arranged at substantially equalintervals, in the above-mentioned thirtieth aspect of the invention.

According to this construction, since the fluorescence of a fluorescentbiomolecule can be observed in the any of the nano-apertures among twoor more nano-apertures, the positioning of said fluorescence detectingmeans can be easily performed.

Moreover, in the case where the interval of the nano-aperture is set thesame as a resolution of a fluorescence detecting means or larger thanthe resolution of a fluorescence detecting means, the fluorescence ofeach fluorescent biomolecule excited by each nano-aperture is separatedso that an interaction between biomolecules can be detected at the levelof a single molecule.

According to a thirty-third aspect of the invention, the presentinvention provides the nano-aperture film, wherein a maximum openingwidth of the nano-aperture is 200 nm or less, in the above-mentionedthirtieth aspect of the invention.

Thus, the maximum opening width of the nano-aperture can be set smallerthan the wavelength of the excitation light.

In a device for analyzing a biomolecular interaction according to athirty-fourth aspect of the invention, the present invention providesthe device for analyzing a biomolecular interaction comprising:

-   -   an excitation light generating means for generating excitation        light;    -   a nano-aperture film which comprises a thin film which does not        transmit light and in which at least one nano-aperture is        formed, wherein a maximum opening width of the nano-aperture is        smaller than the wavelength of the excitation light; and    -   a fluorescence detecting means for detecting fluorescence.

According to this construction, the nano-aperture of the nano-aperturefilm, with a maximum opening width smaller than the wavelength of theexcitation light, can be irradiated with excitation light from theexcitation light generating means, and the evanescent field generated inthe nano-apertures can be used to irradiate the fluorescent biomoleculewith the excitation light in an area smaller than the wavelength of theexcitation light, and the fluorescence emitted from the fluorescentbiomolecule can be detected with the fluorescence detecting means.Moreover, by irradiating the fluorescent biomolecule with the excitationlight in an area smaller than the wavelength of the excitation light,the concentration in the aqueous solution including the fluorescentbiomolecule may be increased. Furthermore, the influence of thenonspecific absorption of the fluorescent biomolecule in a surface ofthe plate such as a glass surface is able to be prevented, and hencedetection or determination of the biomolecular interaction can beperformed reliably.

According to a thirty-fifth aspect of the invention, the presentinvention provides the device for analyzing a biomolecular interaction,wherein a plurality of nano-apertures are provided and arranged at equalintervals, and the interval between the nano-apertures is the same asthe resolution of the fluorescence detecting means or larger than theresolution of the fluorescence detecting means, in the above-mentionedthirty-fourth aspect of the invention.

According to this construction, since the fluorescence of thefluorescent biomolecule is observable in the arbitrary nano-apertures ofa plurality of nano-apertures, alignment by a fluorescence detectingmeans is easy. Moreover, since the interval between the nano-aperturesis the same as the resolution of the fluorescence detecting means orlarger than the resolution of the fluorescence detecting means, thefluorescence of each fluorescent biomolecule excited by eachnano-aperture can be separated, and the interaction between biomoleculescan be detected at the level of a single molecule.

In a method of analyzing a biomolecular interaction according to athirty-sixth aspect of the invention, the present invention provides themethod of analyzing a biomolecular interaction comprising the steps of:

-   -   generating an evanescent field by an excitation light from a        nano-aperture smaller than a wavelength of the excitation light;    -   exciting a fluorescent biomolecule which passes through a        certain region of the evanescent field by Brownian motion; and    -   detecting fluorescence of the fluorescent biomolecule.

Thus, the fluorescent biomolecule in a region smaller than thewavelength of the excitation light can be irradiated with the excitationlight, and the interaction between biomolecules can be detected at thelevel of a single molecule.

In a method of analyzing a biomolecular interaction according to athirty-seventh aspect of the invention, the present invention providesthe method of analyzing a biomolecular interaction comprising the stepsof:

-   -   generating an evanescent field by an excitation light from a        nano-aperture smaller than a wavelength of the excitation light;    -   exciting a first fluorescent biomolecule allowed to attach to        the nano-aperture, and a second fluorescent biomolecule which is        in a certain region of the evanescent field and interacts to the        first fluorescent biomolecule; and    -   detecting fluorescence of these first and second fluorescent        biomolecules, respectively.

Thus, the fluorescent biomolecule in a region smaller than thewavelength of the excitation light can be irradiated with the excitationlight, and the interaction between biomolecules can be detected at thelevel of a single molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of a micro-system showing a first embodiment of thepresent invention, and a sectional view along line A-A thereof.

FIG. 2 is a sectional view of a liquid channel along line B-B of FIG. 1according to the first embodiment of the present invention.

FIG. 3 is a sectional view of the liquid channel along line B-B of FIG.1 according to the first embodiment of the present invention.

FIG. 4 is a sectional view of the liquid channel along line B-B of FIG.1 on a micro-system showing a second embodiment of the presentinvention.

FIG. 5 is a top view of a micro-system showing a third embodiment of thepresent invention.

FIG. 6 is a sectional view of a liquid channel along line C-C of FIG. 5according to the third embodiment of the present invention.

FIG. 7 is a sectional view of the liquid channel along line C-C of FIG.5 showing a modified example according to the third embodiment of thepresent invention.

FIG. 8 is a top view of a micro-system showing a fourth embodiment ofthe present invention.

FIG. 9 is a top view of the micro-system showing a modified exampleaccording to the fourth embodiment of the present invention.

FIG. 10 is a top view of the micro-system showing another modifiedexample according to the fourth embodiment of the present invention.

FIG. 11 is a top view of a micro-system showing a fifth embodiment ofthe present invention.

FIG. 12 is a top view of a micro-system showing a sixth embodiment ofthe present invention.

FIG. 13 is a plain view of a matrix type variable liquid channel showinga seventh embodiment of the present invention.

FIG. 14 is a sectional view of the matrix type variable liquid channelalong line E-E of FIG. 13 according to the seventh embodiment of thepresent invention.

FIG. 15 is a plan view showing a state where the matrix type variableliquid channel is embedded in a basic stand according to the seventhembodiment of the present invention.

FIG. 16 is a schematic diagram of a stimulation detecting means forapplying stimulation by voltage application, according to the seventhembodiment of the present invention.

FIG. 17 is a schematic diagram showing a state where a sample flowsthrough a matrix type variable liquid channel, according to the seventhembodiment of the present invention.

FIG. 18 is a schematic diagram of a system for a matrix type variableliquid channel showing an eighth embodiment of the present invention.

FIG. 19 is a schematic diagram of the matrix type variable liquidchannel showing a state of sample mass movement in an applicationexample 1 according to the eighth embodiment of the present invention.

FIG. 20 is a schematic diagram of the matrix type variable liquidchannel showing a state of sample mass movement in an applicationexample 2 according to the eighth embodiment of the present invention.

FIG. 21 is a schematic diagram of a matrix type variable liquid channelsystem showing a state after sample mass movement in the applicationexample 2 according to the eighth embodiment of the present invention.

FIG. 22 is a schematic diagram of a matrix type variable liquid channelsystem showing a state in which a sample moves in an application example3 according to the eighth embodiment of the present invention.

FIG. 23 is a plain view showing a state in which a sample is surroundedby a wall in the application example 3 according to the eighthembodiment of the present invention.

FIG. 24 is a plain view showing a state in which a liquid channel hasbeen transformed in the application example 3 according to the eighthembodiment of the present invention.

FIG. 25 is a schematic diagram showing a state in which a sample flowsthrough a matrix type variable liquid channel in an application example4 according to the eighth embodiment of the present invention.

FIG. 26 is a figure showing a state in which electrocataphoresis iscarried out in the application example 4 according to the eighthembodiment of the present invention.

FIG. 27 is a front view of a nano-aperture film showing a ninthembodiment of the present invention.

FIG. 28 is a schematic diagram showing the generation of an evanescentfield through nano-apertures according to the ninth embodiment of thepresent invention.

FIG. 29 is a schematic diagram of a measurement principle of FCS usingnano-apertures in a device for analyzing biomolecular interaction,showing a tenth embodiment of the present invention.

FIG. 30 is a schematic diagram of a FCS device with a nano-apertureaccording to the tenth embodiment of the present invention.

FIG. 31 is a schematic diagram of a measurement principle of FCCS usingnano-apertures in a device for analyzing biomolecular interaction,showing an eleventh embodiment of the present invention.

FIG. 32 is a schematic diagram of a FCCS device with a nano-apertureaccording to the eleventh embodiment of the present invention.

FIG. 33 is a schematic diagram of the measurement principle of a singlefluorescent molecule imaging method using the nano-apertures of thedevice for analyzing a biomolecular interaction, showing a twelfthembodiment of the present invention.

FIG. 34 is a schematic diagram of a single fluorescent molecule imagingdevice using the nano-apertures according to the twelfth embodiment ofthe present invention.

FIG. 35 is a schematic diagram of a biomolecule observing method whichuses the conventional evanescent field with a total reflection.

FIG. 36 is a schematic diagram showing a conventional principle of FCS.

DESCRIPTION OF PREFERRED EMBODIMENTS

Description will now be directed to a micro-system, a nano-aperturefilm, a device for analyzing interaction between biomolecules, and amethod of analyzing interaction between biomolecules, according toembodiments of the present invention with reference to the attacheddrawings.

Embodiment 1

Hereafter, the present invention is explained in detail. First, based onFIG. 1 and FIG. 2, a micro-system in a first embodiment of the presentinvention is explained. Numeral 1 denotes a plate consisting of glass,silicone, and the like. The size of the plate 1 is about 10 mm on itsside. A liquid channel 2 is formed on this plate 1. The width of asection of this liquid channel 2 is about 30 μm and the depth is about 5μm. This liquid channel 2 comprises: a liquid channel 2 a; and twoliquid channels 2 b and 2 c branching from the liquid channel 2 a at abranch point 3. Through passages 4 a, 4 b, and 4 c are each formed bypenetrating through the plate 1 from its top to its bottom. The throughpassages 4 a, 4 b, and 4 c are located on the opposite sides of thebranch point 3 to the liquid channels 2 a, 2 b, and 2 c respectively. Acover component 1 a made of glass, etc. is placed closely on the uppersurface of the plate 1, covering the liquid channels 2 a, 2 b, and 2 cand the through passages 4 a, 4 b, and 4 c.

Each of micro-heaters 5 b, 5 c that are heat sources serving as astimulation applying means are provided at the bottom and side of theliquid channels 2 b, 2 c in a portion adjacent to the branch point 3.These micro-heaters 5 b and 5 c are electrically connected to positiveelectrodes 6 b and 6 c respectively, and connected to a negativeelectrode 7. The negative electrode 7 is grounded. End sides of switches8 b and 8 c are connected to the positive electrodes 6 b and 6 crespectively. The other end side of the switches 8 b and 8 c areconnected to a direct-current power supply 10. The switches 8 b and 8 c,and a variable resistor 9 are configured so that operation may becontrolled by a control means not shown in the figure.

As shown in FIG. 3, each of thermo-couples 11 b and 11 c that are heatsensors serving as a stimulation detecting means are provided next tothe micro-heaters 5 b and 5 c at the bottom and side of the liquidchannels 2 b and 2 c in a portion adjacent to the branch point 3. Thesethermo-couples 11 b, 11 c are electrically connected to the controlmeans. The control means controls the voltage applied to themicro-heaters 5 b, 5 c by controlling the variable resistor 9 based on asignal from these thermo-couples 11 b, 11 c.

Next an operation of the invention is explained below. First, liquid forflowing through the liquid channel 2 is introduced from the throughpassage 4 a using a syringe pump (not shown), etc. A heat reversiblehydro-gel material is applied into this liquid. The heat reversiblehydro-gel material causes sol-gel transition at 37 degrees C. At lessthan 37 degrees C. it becomes a sol, and at more than 37 degrees C. itbecomes a gel. A material completely having a heat reversibilityproperty related with change in temperature is preferred for the heatreversible hydro-gel material. For example the material disclosed inJapanese Publication Patent No. H05-262882 can be used. When thetemperature for sol-gel transition is too low, it is not preferredbecause it becomes a gel at room temperature. When too high, it is alsonot preferred because a sample such as protein contained in the liquidbecomes heat denatured during gelation. The temperature for sol-geltransition may be accordingly changed to an appropriate temperature bychoosing the heat reversible hydro-gel material to be used. Also thetype, concentration, etc. of the heat reversible hydro-gel material tobe used can be chosen and adjusted so that it may not react with asample included in the liquid passing through the liquid channel 2, andit may not affect it.

When for example the switch 8 b is turned on by the control means, thevoltage is applied to the micro-heater 5 b formed in the liquid channel2 b to heat the liquid on the micro-heater 5 b. This heat causes theheat reversible hydro-gel material contained in the liquid todrastically turn into a gel. Thereafter the gelled heat reversiblehydro-gel material blocks the portion adjacent to the branch point 3 ofthe liquid channel 2 b. Therefore the liquid flows through the liquidchannel 2 c. At this time, the control means controls the variableresistor 9 based on the signal from the thermo-couple 11 b adjacent tothe micro-heater 5 b to apply heat for an appropriate amount ofstimulation to the liquid. Therefore, there is no possibility that theheat reversible hydro-gel material does not gelate because the amount ofheat applied to the liquid is too small. There is also no possibilitythat the sample contained in the liquid is heat denatured because theamount of the heat applied to the liquid is too large. On the otherhand, if the amount of heat given to the liquid is too large, it maytake much time for the heat reversible hydro-gel material to gelateafter the voltage applied to the micro-heaters 6 b is stopped, andtherefore this is not preferred.

When the switch 8 b is turned of and the switch 8 c is turned on, thevoltage applied to the micro-heaters 5 b is stopped, the liquid on themicro-heaters 5 b becomes cold to solate, and a voltage is applied tothe micro-heaters 5 c formed on the liquid channel 2 c. Thereafter byheating the liquid on the micro-heater 5 c, the gelled heat reversiblehydro-gel material blocks the portion adjacent to the branch point 3 ofthe liquid channel 2 c. Therefore the liquid flows through the liquidchannel 2 b. At this time, the control means controls the variableresistor 9 based on the signal from a thermo-couple 11 c adjacent to themicro-heater 5 c to apply an appropriate amount of the heat for thestimulation to the liquid.

The more minute the liquid channel 2 is, the more quickly the sol-geltransition occurs by the heat reversible hydro-gel material. It ispossible to switch the sol-gel transition by the millisecond when thewidth of a section of the liquid channel 2 is about 30 μm and the depthis about 5 μm, as with the embodiment in this invention. Therefore, theliquid channels 2 b and 2 c can be changed very quickly, and hence itbecomes possible to certainly isolate the required sample in the liquidby using this.

As mentioned above, according to this embodiment, a micro-system has themicro-heaters 5 b and 5 c for serving as the stimulation applying meansfor applying stimulation to the liquid flowing through the liquidchannels 2 b and 2 c formed on the plate 1, the liquid flow beingcontrolled by the stimulation from the micro-heaters 5 b and 5 c. Themicro-system comprises a control means for controlling an amount ofstimulation applied to the liquid by the micro-heaters 5 b and 5 c. Thusthis control means can provide an appropriate amount of stimulation byelectrically controlling an amount of stimulation applied to the liquidfrom the micro-heaters 5 b and 5 c.

The micro-system also has thermo-couples 11 b and 11 c, serving asstimulation detecting means for detecting an amount of stimulation, andthe control means controls the micro-heaters 5 b and 5 c based on asignal from the thermo-couples 11 b and 11 c. Thus an appropriate amountof stimulation can be provided. By using the micro-heaters 5 b and 5 c,stimulation can be certainly applied to liquid.

Also by using the thermo-couples 11 b and 11 c, the amount ofstimulation applied to the liquid by the micro-heaters 5 b and 5 c canbe reliably detected, which enables a heat sensor to be easilyconstructed.

Embodiment 2

Next a second embodiment of the present invention is explained. The samereference symbols are given to the same portions as in the above firstembodiment, and detailed explanation is omitted. In this embodiment, asshown in FIG. 4, heat sensitive semiconductors 12 b and 12 c areprovided for serving as a stimulation detecting means instead of thethermo-couples 11 b and 11 c in the first embodiment. These heatsensitive semiconductors 12 b and 12 c are formed in the cover component1 a above the micro-heaters 5 b and 5 d. Also instead of these heatsensitive semiconductors 12 b and 12 c, an infrared ray sensitive sensormay be provided.

As mentioned above, according to this embodiment, since the heatsensitive semiconductors 12 b and 12 c or an infrared ray sensitivesensor are used, the amount of stimulation can be reliably detected.

Embodiment 3

Next, a third embodiment of the present invention is explained. In FIG.5 and FIG. 6, numeral 21 denotes a plate which comprises a transparentmaterial, such as glass. The size of the plate 21 is about 10 mm on itsside. A liquid channel 22 is formed on this plate 21. The width of asection of this liquid channel 22 is about 30 μm and the depth is about5 μm. This liquid channel 22 comprises a liquid channel 22 a, and twoliquid channel 22 b and 22 c branching from this liquid channel 22 a ata branch point 3. Through passages 24 a, 24 b, and 24 c are each formedby penetrating through the plate 21 from its top to its bottom. Thethrough passages 24 a, 24 b, and 24 c are located on the opposite sidesof the branch point 23 to the liquid channels 22 a, 22 b, and 22 crespectively. A cover component 21 a made from a transparent materialsuch as glass is placed closely on the upper surface of the plate 21,covering the liquid channels 22 a, 22 b, and 22 c and the throughpassages 24 a, 24 b, and 24 c.

Semiconductor lasers 25 b, 25 c that are light emitting elements orlight sources serving as a stimulation applying means are provided nextto the liquid channels 22 b, 22 c in a portion adjacent to the branchpoint 23. As shown in FIG. 6, the semiconductor lasers 25 b and 25 c areembedded in the plate 21, and are comprised so that the liquid channels22 b and 22 c in the portion adjacent to the branch point 23 may beirradiated with the infrared laser light from each light-emitting part26 b and 26 c of the semiconductor lasers 25 b and 25 c. Thesemiconductor lasers 25 b and 25 c are constituted so that a controlmeans not shown in the figure can control their operation. As shown inFIG. 7, the semiconductor lasers 25 b and 25 c may be formed byembedding them between the plate 21 and the cover component 21 a.

Moreover, the infrared ray sensitive sensors (not shown) arerespectively provided in the area irradiated with the infrared laserlight using the semiconductor lasers 25 b and 25 c of the liquidchannels 22 b and 22 c. The infrared ray sensitive sensors areelectrically connected to the control means, and the control means isconstructed so that it can control the operation of the semiconductorlasers 25 b and 25 c based on the signal from the infrared ray sensitivesensors.

Next the operation is explained below. Firstly a liquid for flowingthrough the liquid channel 22 is introduced from the through passage 24a using a syringe pump (not shown), or the like. A heat reversiblehydro-gel material is added to this liquid. The heat reversiblehydro-gel material causes sol-gel transition at 37 degrees C. At lessthan 37 degrees C. it becomes a sol, and more than 37 degrees C. itbecomes a gel. Since the heat reversible hydro-gel material is the sameas that used in the first embodiment, detailed explanation is omitted.

Using the control means, for example, when the semiconductor laser 25 bis turned on, the liquid in the portion adjacent to the branch point 23of the liquid channel 22 b is irradiated with infrared rays of laserlight from the semiconductor laser 25 b to heat the liquid in theportion. This heat causes the heat reversible hydro-gel materialcontained in the liquid to drastically turn to a gel. Thereafter thegelled heat reversible hydro-gel material blocks the portion adjacent tothe branch point 23 of the liquid channel 22 b. Therefore the liquidflows through the liquid channel 22 c. At this time the control meanscontrols the semiconductor laser 25 b based on the signal from theinfrared ray sensitive sensor to apply the infrared laser light as anappropriate amount of stimulation to the liquid. Therefore, there is nopossibility that the heat reversible hydro-gel material does not turn toa gel when the infrared laser light applied to the liquid is too small.There is also no possibility that the sample contained in the liquid isheat denatured since the infrared laser light applied to the liquid istoo large.

When the semiconductor laser 25 b is turned off and the semiconductorlaser 25 c is turned on, the liquid in the portion adjacent to thebranch point 23 of the liquid channel 22 b where the infrared laserlight having been irradiated until then, gets colds to gelate.Thereafter the infrared laser light is applied to the liquid in theportion adjacent to the branch point 23 of the liquid channel 22 c fromthe semiconductor laser 25 c to heat it. This heat causes the heatreversible hydro-gel material contained in the liquid to drasticallyturn to a gel. Thereafter the gelled heat reversible hydro-gel materialblocks the portion adjacent to the branch point 23 of the liquid channel22 c. Therefore the liquid flows through the liquid channel 22 b. Atthis time, the control means controls the semiconductor laser 25 c basedon the signal from the infrared ray sensitive sensor to apply theinfrared laser light as an appropriate stimulation to the liquid.

As mentioned above, according to this embodiment, a micro-systemcomprises the semiconductor lasers 25 b and 25 c being a stimulationapplying means for applying stimulation to a liquid flowing in theliquid channels 22 b and 22 c formed in the plate 21, the liquid flowbeing controlled by the stimulation from the stimulation applying means,wherein the semiconductor lasers 25 b and 25 c comprise a control meansfor electrically controlling an amount of stimulation applied to theliquid. By electrically controlling the amount of stimulation applied tothe liquid by the semiconductor lasers 25 b and 25 c using the controlmeans, it is possible to give an appropriate amount of stimulation.

Also a micro-system comprises an infrared ray sensitive sensor servingas a stimulation detecting means, for detecting the amount ofstimulation, and said semiconductor lasers 25 b and 25 c are controlledby said control means based on a signal from said infrared ray sensitivesensor. Using the semiconductor lasers 25 b and 25 c serving as thelight emitting element, it is possible to reliably give stimulation tothe liquid.

Moreover, by using the infrared ray sensitive sensor, it is possible toreliably detect the amount of stimulation applied to the liquid by thesemiconductor lasers 25 b and 25 c.

Furthermore, because the semiconductor lasers 25 b and 25 c are providedin the plate 21, it is possible to easily construct a light source.

Also because the semiconductor lasers 25 b and 25 c are embedded in theplate 21, it is possible to arrange the semiconductor lasers 25 b and 25c near the liquid channels 22 b and 22 c, and to reliably givestimulation to the liquid.

Embodiment 4

Next, a fourth embodiment of the present invention is explained. Thesame reference symbols are given to the same portions as in the abovethird embodiment, and detailed explanation is omitted. In thisembodiment, as shown in FIG. 8, a plate 21 is mounted on a stand 31, andsemiconductor lasers 25 b and 25 c are arranged on the stand 31 outsidethe plate 21. In this embodiment, as shown in FIG. 9, opticalintroducing paths 27 b and 27 c may be provided for guiding infraredlaser light emitted from the semiconductor lasers 25 b and 25 c toliquid channels 22 b and 22 c. These optical guiding paths 27 b and 27 care formed horizontally with the surface of the plate 21 in which theliquid channels 22 b and 22 c are formed. These optical guiding paths 27b and 27 c are provided by forming cavities in the plate 21, and placingmetal foil on that side for reflecting infrared laser light. Or thecavity may be filled with a material whose refractive index is lowerthan the material forming the plate 21 for inducing total internalreflection of the infrared laser light on the side of the cavity.Conversely, the cavity may be configured so as to be filled with amaterial whose refractive index is higher than the material forming theplate 21 for inducing total internal reflection of the infrared laserlight on the side of the cavity.

As mentioned above, according to this embodiment, the semiconductorlasers 25 b and 25 c are arranged outside the plate 21. The plate 21 maybe used as a disposable one, and the semiconductor lasers 25 b and 25 cmay be repeatedly used.

The optical guiding paths 27 b and 27 c for guiding light from thesemiconductor lasers 25 b and 25 c are formed horizontally with thesurface of the plate 21 in which the liquid channels 22 b and 22 c areformed. It is possible to efficiently guide the light from thesemiconductor lasers 25 b and 25 c to the liquid channels 22 b and 22 c.

Since the micro-system comprises a plurality of light emitting elements,it is possible to apply the stimulation at a number of different sitesin the liquid channels 22 b and 22 c.

As shown in FIG. 10, by controlling mirrors 28 b and 28 c by a controlmeans not shown in the figure, the infrared laser light from onesemiconductor laser 25 may be selectively emitted to either of the twoliquid channels 22 b and 22 c.

Embodiment 5

Next, a fifth embodiment of the present invention is explained. The samereference symbols are given to the same portions as in the above thirdand fourth embodiments, and detailed explanation thereof is omitted. Inthis embodiment, as shown in FIG. 11, a semiconductor laser 41 being anenergy imparting means for imparting excitation light as energy toliquid in a liquid channel 22 a; and a fluorescence detecting element 42being a change detecting means for detecting fluorescence from asubstance excited by the excitation light from this semiconductor laser41, are arranged on a mounting stand 31 outside of the plate 21 Thefluorescence detecting element 42 is configured so as to switch to anexcitation light detecting element 42 a for detecting the excitationlight from the semiconductor laser 41 as necessary. As mentionedhereinafter, this excitation light detecting element 42 a acts as apositioning means for deciding a position of the plate 21 on the stand31. Instead of the fluorescence detecting element 42, a light receivingelement may be configured as the change detecting means.

An energy introducing path 43 for guiding excitation light emitted fromthe semiconductor laser 41 to the liquid channels 22 a is formed on theplate 21. This energy guiding path 43 is arranged horizontally with thesurface of the plate 21 in which the liquid channel 22 a is formed. Thisenergy introducing path 43 is provided by forming a cavity in the plate21, and placing metal foil on that side for reflecting excitation light.Or the cavity may be configured so as to be filled with a material whoserefractive index is lower than the material forming the plate 21 forinducing total internal reflection of the excitation light on the sideof the cavity. Conversely, the cavity may be configured so as to befilled with a material whose refractive index is higher than thematerial forming the plate 21 for inducing total internal reflection ofthe excitation light on the side of the cavity.

Furthermore a fluorescence guiding path 44 for guiding the fluorescenceemitted from the substance in the liquid of the liquid channel 22 a to afluorescence detecting element 42 is provided on the plate 21. Thefluorescence is excited by excitation light from the semiconductor laser41. This fluorescence guiding path 44 is formed horizontally with thesurface of the plate 21 in which the liquid channel 22 a is formed. Aswith the above energy introducing path 43, this fluorescence guidingpath 44 is provided by forming a cavity in the plate 21, and placingmetal foil on that side for reflecting excitation light. Or the cavitymay be configured so as to be filled with a material whose refractiveindex is lower than the material forming the plate 21 for inducing totalinternal reflection of the excitation light on the side of the cavity.Conversely, the cavity may be configured so as to be filled with amaterial whose refractive index is higher than the material forming theplate 21 for inducing total internal reflection of the excitation lighton the side of the cavity. The energy introducing path 43 is formed inalignment with the fluorescence guiding path 44.

The semiconductor laser 41 is controlled by the control means not shownin the figure to control the semiconductor lasers 25 b and 25 c based onthe signal from the fluorescence detecting element 42.

Next the operation is explained. First the plate 21 is mounted on thestand 31. At this time the plate 21 is fixed at a position where theexcitation light from the semiconductor laser 41 via the energyintroducing path 43 and the fluorescence guiding path 44 can be detectedmost strongly in the excitation light detecting element 42 a. Thus byusing the semiconductor laser 41 and the excitation light detectingelement 42 a as a positioning means like this, it is possible toprecisely mount the plate 21 in a predetermined position on the stand 31when replacing the plate 21.

The liquid for flowing through the liquid channel 22 is introduced fromthrough passage 24 a using a syringe pump not shown in the figure or thelike. A heat reversible hydro-gel material is added to this liquid. Theheat reversible hydro-gel material causes sol-gel transition at 37degrees C. At less than 37 degrees C. it becomes a sol, and at more than37 degrees C. it becomes a gel. Because the heat reversible hydro-gelmaterial is the same as that used in the first embodiment, detailedexplanation is omitted.

Hereinafter a case of sorting a sample in this liquid is described as anexample. The sample may be, for example, protein molecule, and belabeled with a fluorescent substance if necessary. The speed forintroducing the liquid from the syringe pump, or the like, is adjustedbeforehand so that the liquid may flow into the liquid channel 22 a atabout 2 mm/second. For the speed of flow, 100 mm/second or less issuitable. However, the speed of flow is a value which is decided by adetector configuration and a channel structure, and is not essential forthis invention. Excitation light is emitted to the liquid flowingthrough the liquid channel 22 a, from the semiconductor laser 41 throughthe energy introducing path 43. Thereafter the fluorescence from atarget sample through the fluorescence guiding path 44 is detected bythe fluorescence detecting element 42. The fluorescence is detected bythis fluorescence detecting element 42, for example, for every 10 ms,and the result is outputted to the control means. However, the cycle fordetecting the fluorescence is a value which is not decided by thedetector configuration, and is not essential for the invention.

When the fluorescence is not detected from the target sample, thecontrol means turns on the semiconductor laser 25 b, and the infraredlaser light is emitted from the semiconductor laser 25 b to the liquidin the portion adjacent to the branch point 23 of the liquid channel 22b to heat it. By this heating, the heat reversible hydro-gel materialincluded in the liquid drastically turns into a gel, and this gelledheat reversible hydro-gel material blocks the part near the branch point23 of the liquid channel 22 b. Therefore, the liquid flows into the sideof the liquid channel 22 c, and this liquid is discarded from thethrough passage 24 c. At this time, the control means controls thesemiconductor laser 25 b based on a signal from the infrared raysensitive sensor to apply the infrared laser light being an appropriateamount of stimulation to the liquid.

When fluorescence is detected from the target sample, the control meansturns off the semiconductor laser 25 b and turns on the semiconductorlaser 25 c. Thereafter the liquid in the portion adjacent to the branchpoint 23 of the liquid channel 22 b, the liquid having been irradiatedwith the infrared laser light by then, gets cold to drastically solate.The infrared laser light is emitted from the semiconductor laser 25 c tothe liquid in the portion adjacent to the branch point 23 of the liquidchannel 22 c to heat it. By this heating, the heat reversible hydro-gelmaterial included in the liquid drastically turns into a gel, and thisgelled heat reversible hydro-gel material blocks the part near thebranch point 23 of the liquid channel 22 c. Therefore, the liquid flowsinto the side of the liquid channel 22 b, and this liquid is collectedfrom the through passage 24 b. At this time, the control means controlsthe semiconductor laser 25 c based on the signal from the infrared raysensitive sensor to apply the infrared laser light being the appropriateamount of stimulation to the liquid.

Thus the target sample can be isolated in units of one molecule byirradiating the liquid flowing through the liquid channel 22 b with theinfrared laser light from the semiconductor laser 41, detecting thefluorescence from the target sample by the fluorescence detectingelement 42 with a very short period of every 10 ms, and furthercontrolling the semiconductor lasers 25 b and 25 c by the control meansbased on this detection result to switch the liquid flow to the liquidchannel 22 b or the liquid channel 22 c.

As mentioned above according to this embodiment, the micro-systemfurther comprises: the semiconductor laser 41 being the energy impartingmeans for imparting the excitation light as energy to the liquid; andthe fluorescence detecting element 42 being the change detecting meansfor detecting the fluorescence of the substance producing thefluorescence by the excitation light from the semiconductor laser 41,wherein the semiconductor lasers 25 b and 25 c are controlled by thecontrol means based on a signal from the fluorescence detecting element42. By controlling the liquid flow based on the substance producing thefluorescence by the excitation light from the semiconductor laser 41, itis possible to easily separate only the liquid containing the substance.

The energy guiding path 43 for guiding the excitation light from thesemiconductor laser 41 is formed horizontally with the surface of theplate 21. Thus, it is possible to efficiently guide the excitation lightfrom the semiconductor laser 41 to the liquid channel 22 a.

By using the fluorescence detecting element 44 or the light receivingelement, it is possible to certainly detect the fluorescence of thesubstance producing the fluorescence by the excitation light from thesemiconductor laser 41.

The fluorescence detecting element 44 or the light receiving element isarranged horizontally with the surface of the plate 21. Thus, it ispossible to detect the fluorescence of the substance producing thefluorescence by the excitation light from the semiconductor laser 41from the sides of the liquid channel 22 a.

Moreover, the micro-system comprises: the stand 31 for mounting theplate 21; and the semiconductor laser 41 and excitation light detectingelement 42 a being the positioning means for deciding the position ofthe plate 21 on the stand 31. By the semiconductor laser 41 and theexcitation light detecting element 42 a, it is possible to easily mountthe plate 21 in the correct position of the stand 31. In particular,when the plate is used as a disposable one, the effort of positioningwhen mounting the plate 21 correctly can be saved.

Embodiment 6

Next a sixth embodiment of the present invention is explained. The samereference symbols are given to the same portions as in the above fifthembodiment, and detailed explanation thereof is omitted. In thisembodiment, as shown in FIG. 12, the semiconductor laser 41 is embeddednear the liquid channel 22 a, and the fluorescence detecting element 44is arranged above the part irradiated with the excitation light in theliquid channel 22 a. Instead of the fluorescence detecting element 44,the fluorescence detecting element may be arranged above the partirradiated with the excitation light in the liquid channel 22 a.

Guide components are arranged at three points as the positioning meansfor fixing the plate 21 in a predetermined position on the stand 31.These guide materials 51 for setting three corners of the plate 21, areformed together with the stand 31. Instead of providing the guidematerial 51, the micro-system may be configured so as to decide theposition of the plate 21 on the stand 31 by making a marking 52 and amarking 53 on the plate 21 and the stand 31 respectively, and aligningthe marking 52 with the marking 53. Also the micro-system may beconfigured so as to decide the direction of the plate 21 as well as theposition by using both the guide material 51 and the markings 52 and 53.Furthermore the micro-system may be configured so as to decide theposition of the plate 21 on the stand 31 by forming a concave part and acorresponding convex part at the bottom of the plate 21 and on the uppersurface of the stand 31 respectively, and fitting the convex part intothe concave part.

As mentioned above, according to this embodiment, the fluorescencedetecting element 42 or the light receiving element is arranged abovethe liquid channel 22 a. Therefore the fluorescence of the substanceproducing the fluorescence by the excitation light from thesemiconductor laser 41 can be detected from above the liquid channel 22a.

The micro-system, further comprises: the stand 31 for mounting the plate21; and the guide material 51 being the positioning means for decidingthe position of the plate 21 on the stand 31. It is possible to easilymount the plate 21 in the correct position of the stand 31 by the guidematerial 51. In particular, when the plate is used as a disposable one,the effort of positioning when mounting the plate 21 correctly can besaved.

This invention is not limited to the above embodiment. Many othervariations are possible within the scope of this invention. For example,the liquid channel formed on the plate may branch into more than three,or flow together. If necessary, the position and number of stimulationapplying means, stimulation detecting means, and change detecting meansmay be appropriately changed. Moreover, the stimulation applied by thestimulation applying means may be voltage, and a voltage reversiblehydro-gel material causing sol-gel transition in proportion to the riseand fall of the voltage may be used. Furthermore, the micro-system maybe constructed by combining a plurality of plates.

Embodiment 7

Next, based on FIG. 13 and FIG. 14, a matrix type variable liquidchannel being a seventh embodiment of the present invention isexplained.

A matrix type variable liquid channel is configured so that metal pieces103 being the stimulation sensitive members are arranged on a glassplate 102 at an interval of 10 μm in each direction in a pattern of atwo dimensional matrix, that is to say, as a two dimensional matrix. Theheight of the glass plate 102 is 200 μm, the width is 200 μm, and thethickness is 5 μm. The height of the metal pieces 103 is 10 μm, thewidth is 10 μm, and thickness is 6 nm.

The shape of these stimulation sensitive members is not limited to aflat square, and the shape may be a rectangle, a polygon, or a circular.

The metal pieces 103 being stimulation sensitive members can be formedby a usual method such as a masking method, by vapor deposition,sputtering, chemical vapor deposition (CVD), plating, plasmapolymerization, or screen-printing of metal such as titanium, chromium,and the like.

A plurality of external connection channels 104 having a width of 20 μmand a depth of 5 μm are provided on the four sides of the glass plate102. On two of these sides, four external connection channels 104 arearranged, with inlets 104 a facing outlets 104 b. On the other two ofthe four sides, four external connection channels 104 are arranged, withinlets 104 c facing outlet 104 d.

The glass plate 102 (matrix type variable liquid channel) is set in acentral part of a basic stand 101, as shown in FIG. 15. The size of thisbasic stand 101 is about 20 mm on one side. This basic stand 101consists of glass, silicone, or the like.

The basic stand 101 and the glass plate 102 need not always be separate,and they may be configured so as to be united with each other. Forexample, as mentioned below, in the case of applying the stimulation tothe stimulation sensitive members 103 using light, the stimulationsensitive members 103 may be irradiated with light from a side of thebasic stand 101. The basic stand 101 and the glass plate 102 may beconfigured so as to be united with each other using glass, or the like.On the other hand, when applying the stimulation to the stimulationsensitive members 103 using a switch controller, it is better toconstruct them so as to be separate from each other, because it isconvenient to embed an element such as diode between the basic stand 101and the glass plate 102.

The external connection channels 104 connected to the glass plate 102are provided in this basic stand 101. Through passages 105 penetratingthe basic stand 101 are formed in the opposite side to the glass plate102. A solution can be flowed in from the through passages 105 into theexternal connection channels 104 using a syringe pump not shown in thefigure. The solution flowing in the glass plate 102 can be flowed outthrough the external connection channels 104 to the through passages105.

There is provided a cover glass 108 on the basic stand 101. Thethickness of the cover glass 108 is 100 μm so as to cover the glassplate 102 and the external connection channel 104 completely. Thus thesolution in the glass plate flows through an area between the glassplate 102 and the cover glass 108. An interval (height) between thisglass plate 102 and the cover glass 108 is preferably 5 to 20 μm.

In FIG. 15, the through passages 105 are provided in the basic stand101, but the configuration is not particularly limited to this. Thethrough passages 105 may be provided in a position which does notobstruct sample detection, for example on the side of the cover glass108 or the basic stand 101.

As a method of apply stimulation to the metal pieces 103 being thestimulation sensitive members, the following methods can be used: amethod of applying a voltage to the metal piece 103 by a switchcontroller to heat it; a method of irradiating the metal piece 103 withlaser light using a scanner mirror, or an acousto-optic deflector; and amethod of irradiating the metal piece 103 with the laser light or lamplight using a digital mirror device.

FIG. 16 shows a concept of a method of heating the metal piece 103 by aswitch controller. This method involves incorporating a circuit into theglass plate 102, and making the metal piece 103 generate heat by aswitching element 112. The circuit consists of a matrix of the metalpiece 103 as a resistance body (stimulation sensitive members) and adiode 111.

In FIG. 16, a circuit for line i is shown schematically as an example.Similar circuits are provided from line 1 to line m (m is an arbitraryinteger) and from row 1 to row n (n is an arbitrary integer). The metalpieces 103 are formed in the parts where the line intersects with therows. For example when inputting at line i and row j (for examplechanging the voltage to Low), the voltage is applied to the metal piece103 of line i and row j to pass an electric current to generate heat.This input can be controlled by using a computer.

A method of heating the metal piece 103 using light includes thefollowing methods: a method of irradiating the metal piece 10 with laserby using a scanner mirror or an acousto-optic deflector. For example, byusing an infrared ray laser such as an Nd:YAG laser (oscillation wavelength 1064 nm, 800 mW), the metal piece 103 can be heated by inputtingan output from a DA conversion board installed in a computer to theservo driver of the scanner mirror, or, for example, an acousto-opticdeflector N45000 made by the NEOS Technologies company, so that a beammoves along the pattern of the passage. In this case, a laser having anoscillation wave length of about 300 nm to about 1600 nm can be used.Especially a semiconductor laser (infrared ray laser) having anoscillation wave length of about 700 nm to about 1600 nm is preferredbecause it does not prevent detecting a biological sample.

In a method of irradiating the metal piece 103 with a digital mirrordevice, for example, dual monitors are provided in a computer. A firstmonitor is used for both output and operation of an image for analyzingthe image. A pattern of the liquid channel is outputted to a secondmonitor by a program for outputting a pattern of the liquid channel. Theoutput from the second monitor is outputted to a digital mirror deviceas the pattern of the liquid channel. The digital mirror device islocated at a position being optically coupled with the matrix typevariable liquid channel. The metal piece 103 can be heated byirradiating it with light of a laser or a lamp (mercury lamp or xenonlamp).

Next, wall or valve structures forming the liquid channel are explained.

The solution flows out from the external connection channel 104 into theglass plate 102. For example by including a heat sensitive substance inthe solution, and heating the heat sensitive substance for stimulation,the solution can be reversibly turned to a sol or a gel.

As the heat sensitive substance, a heat reversible hydro-gel materialcan be used. The heat reversible hydro-gel material causes sol-geltransition at 37 degrees C. At less than 37 degrees C. it becomes a sol,and at more than 37 degrees C. it becomes a gel. A material havingcomplete reversibility corresponding to change in temperature ispreferred, as the heat reversible hydro-gel material. For example thematerial disclosed in Japanese Publication Patent No. H05-262882 can beused. A preferred material is for example, methyl-cellulose or Mebiolgel (sol-gel transition temperature approximately 36 degrees C.).

When the temperature for sol-gel transition is too low, it is notpreferred because it becomes a gel at room temperature. When too high,it is also not preferred because a sample such as protein contained inthe liquid is heat denatured while turning to a gel. The temperature forsol-gel transition may be accordingly changed to the appropriatetemperature by choosing the heat reversible hydro-gel material to beused.

Also the type, concentration, etc. of the heat reversible hydro-gelmaterial to be used can be chosen and adjusted so that it may not reactwith the solution and the sample included in the solution and it may notaffect it.

The solution flows in the glass plate 102. By heating the metal piece103 on the glass plate 102, the heat sensitive substance included in thesolution turns to a gel by sol-gel transition. The gel becomes the wallor valve structure forming the liquid channel. The method of heating themetal piece 103 on the glass plate 102 is as follows; a method ofheating the metal piece 103 using the above switch controller; a methodof irradiating the metal piece 103 with the infrared laser using ascanner mirror, or an acousto-optic deflector; and a method ofirradiating the metal piece 103 with light using a digital mirrordevice.

When there is a demand for forming the liquid channel before the samplematerial flows in the glass plate 102, the liquid channel can be formedby filling the glass plate 102 with the solution including the heatsensitive substance, and heating the arbitrary metal piece 103 under theabove condition.

FIG. 17 shows an aspect of where stimulation is given to the glass plateshown in FIG. 13, to gel at an arbitrary position and make a wall orvalve structure. The same reference symbols are used for the same partsas in FIG. 13, and the description is omitted.

In FIG. 17, the solution including the heat sensitive substance isflowed in from a first inlet (In1), a second inlet (In2), and a fourthinlet (In4) of the inlet 104 a to the glass plate 102, and an arbitrarymetal piece 103 is heated by the stimulation applying means such asvoltage or light mentioned above. Thus the heat sensitive substanceflowing through on the metal piece 103 is turned into a gel to form thewall 106 on the glass plate 102.

In FIG. 17, numeral 107 denotes the sample included in the solution. Thearrow at the front edge indicates the flow direction of this sample 107.

In FIG. 17, the sample 107 together with the solution is flowed out to afirst outlet (Out1) and a third outlet (Out3) of the outlet to theexternal connection channel 104.

There are cases where a specific amount of the sample 107 flows out fromthe first outlet (Out1) and the third outlet (Out3), or where an othersample flows in on the glass plate 102, and so on. In such cases, ifnecessary, the liquid channel can be changed by turning the wall 106formed by gellation by the aforementioned stimulation applying meansinto a sol, and forming the wall or valve structures 106 by applying thestimulation to the metal piece 103 located in a new part to turn it intoa gel.

In addition there is a case where the desired sample 107 flows out onthe glass plate 102. If the stimulation is applied to the metal piece103 located surrounding the sample 107, the sample 107 can be made tostay within the glass plate 102. Thus the sample can be analyzed.

As mentioned above, according to this invention, the wall or valvestructure can be formed reversibly through the sol-gel transition at anypositions by stimulating a plurality of stimulation sensitive members103 arranged in a pattern of the two dimensional matrix on the glassplate 102. Thus the liquid channel can be easily made. Because channelshape can be freely changed, it is not necessary to prepare liquidchannels having different channel shapes.

Moreover, because the stimulation sensitive members 103 are stimulated,the gelation rate of a substance having sol-gel transition propertiesincreases. In addition since there is an interval between thestimulation sensitive members, gelation at any positions is facilitated.Furthermore, by connecting the gelling area, the wall or valve structure106 can be formed.

The stimulation sensitive members 103 are formed by vapor deposition,sputtering, Chemical Vapor Deposition (CVD), plating, plasmapolymerization, or screen-printing, and thus they can be easily formed.

The stimulation sensitive member 103 is stimulated by applying a voltageor irradiating a light thereto. Thus the temperature of the stimulationsensitive member 103 can be adjusted, and the sol-gel transition can beeasily initiated.

Embodiment 8

Next, an eighth embodiment of the present invention is explained.

FIG. 18 shows a schematic diagram of a matrix type variable liquidchannel system of the present invention.

The matrix type variable liquid channel system comprises: a matrix typevariable liquid channel 121 which comprises a plurality of stimulationsensitive members 103 being arranged on the glass plate 102 in a patternof a two dimensional matrix; a detecting means 122 for detecting asubstance on the glass plate 102; a stimulation applying means 123 forapplying stimulation to the stimulation sensitive members 103; and acontrol means 124 for controlling the stimulation applying means 123based on a signal from the detecting means 122.

For this matrix type variable liquid channel 121, the matrix typevariable liquid channel fitted to the basic stand 101 explained in theseventh embodiment of the invention can be used, and hence itsexplanation is omitted here.

The detecting means 122 for detecting the substance in the matrix typevariable liquid channel 121 on the glass plate 102 is provided with amicroscope 122 b including an object lens 122 a, a detecting device 122c and an analyzing device 122 d.

General sensors such as a video camera, an avalanche photodiode, or aphotoelectron multiplier can be used as the detecting device 122 c. Animage analyzing device and a device for analyzing the detection resultof the general sensors can be used as the analyzing device 122 d. In thecase of using the video camera as the detecting device 122 c, the imageanalyzing device can be used as the analyzing device 122 d. In the caseof using the general sensors as the detecting device 122 c, the devicefor analyzing the detection result of the general sensors can be used asthe analyzing device 122 d.

The stimulation applying means 123 is for applying stimulation to thestimulation sensitive members 103 formed in the matrix type variableliquid channel 121 on the glass plate 102.

As the stimulation applying means 123, the following methods explainedin the seventh embodiment can be used; a method of applying a voltage tothe stimulation sensitive members 103 by a switch controller to heat it;a method of irradiating the stimulation sensitive members 103 with laserlight using a scanner mirror, or an acousto-optic deflector; and amethod of irradiating the stimulation sensitive members 103 with thelaser light or the lamp light using a digital mirror device. Explanationof these is omitted here.

The control means 124 is for controlling the stimulation applying means123, based on a signal from the detecting means 122. The control means124 can control which stimulation sensitive members are irradiated ofthe stimulation sensitive members formed in the matrix type variableliquid channel 121 on the glass plate 102. The control means 124 canalso control the strength, time, etc. of the stimulation.

By means of these configurations, when the solution including the sampleand the heat sensitive substance flows in the matrix type variableliquid channel 121 on the glass plate 102, the flow is captured with theobject lens 122 a. Thereafter, it is guided to an optical microscope 122b, is saved as data in the detecting means 122 c connected to theoptical microscope 122 b, and this data is analyzed using the analyzingdevice 122 d.

Based on the analysis result from this analyzing device 122 d,stimulation is applied to the metal pieces 103 being arbitrarystimulation sensitive members on the glass plate 102 by a switch controlby the control means 124, so that the stimulation is applied to themetal pieces 103 being the arbitrary stimulation sensitive members onthe glass plate 102. This stimulation heats the metal pieces 103, afterwhich the heat sensitive substance turn into a gel through sol-geltransition to form the wall or valve structure 106 on the glass plate102. Thus the channels can be freely formed.

In the case of using an image analyzing device as the analyzing device122 d, for example, the following images are shown: the image of FIG. 13showing the liquid channels before the wall or valve structure 106 isformed on the glass plate 102; and the image of FIG. 17 showing theliquid channels formed with the wall or valve structure 106 on the glassplate 102, and the target sample flowing through these channels.

When confirming that target substance flows through the liquid channels,with the analyzing device 122 d, the stimulation is applied to the metalpiece 103 on the glass plate 102 by the control means 124, andthereafter the heat sensitive substance turn into a gel to form the wallor valve structure 106 at any positions on the glass plate 102. Thus thechannels can be changed, and the target sample 107 can be held on theglass plate 102 by surrounding it with the wall 106.

This control is not limited to visual control, and a control means forautomatic controlling with a computer or the like may be used.

The sample flowing through the liquid channel, or the sample held on theglass plate 102 may be analyzed with the analyzing device 122 dpreviously loaded with analytical functions, or an analyzing device (notshown) connected to the detecting device 122 c.

As mentioned above, according to the embodiment of the presentinvention, the matrix type variable liquid channel system comprises: thematrix type variable liquid channel 121 which comprises a plurality ofstimulation sensitive members 103 arranged on the plate 102 in a patternof a two dimensional matrix; the detecting means 122 for detecting thesubstance on the plate 102; the stimulation applying means 123 forapplying stimulation to the stimulation sensitive members 103; and thecontrol means 124 for controlling the stimulation applying means 123based on a signal from the detecting means 122. The wall or valvestructure 106 can be formed reversibly through a sol-gel transition atthe positions corresponding to the stimulation sensitive member 103, byapplying stimulation to it, and thus the liquid channels can be easilymade. The liquid channels can be easily formed by controlling thestimulation applying means 123. Thus, it is not necessary to prepareliquid channels having different channel shapes. In addition thesubstance can be detected at any positions on the plate 102, and hencethe desired sample substance is easily separated or analyzed.

The stimulation is applied to the stimulation sensitive member 103, andhence the gelation rate of the substance having sol-gel transitionproperties increases. There is an interval between the stimulationsensitive members, and hence it is easier to turn it into a gel at anyposition.

Because the wall or valve structure 106 can be formed by connecting thegelling area, the liquid channels can be easily changed on the glassplate 102, and the target sample can be easily separated or analyzed.

Moreover, because the stimulation sensitive members are formed by vapordeposition, sputtering, Chemical Vapor Deposition (CVD), plating, plasmapolymerization, or screen-printing, they can be easily formed. Thus thematrix type variable liquid channel system can be made cheaply.

The means for applying a voltage or irradiating a light thereto is usedas the stimulation applying means 123. Thus, the stimulation can beeasily applied to the stimulation sensitive members 103, the temperatureof the stimulation sensitive members 103 can be adjusted, and thesol-gel transition can be easily initiated.

Moreover, by such a construction, the liquid channels on the plate canbe easily changed, and hence the substance can be detected at anyposition on the plate, and the target sample can be easily separated oranalyzed.

Next, application examples 1 to 4 in this invention are explained usingFIG. 19 to 26. In the application examples, the same reference symbolsdenote the same portion as in the above seventh embodiment and eighthembodiment, and the detailed explanation is omitted. Moreover, becausethe configuration of the matrix type variable liquid channel system isthe same as the one in the eighth embodiment, its drawing is notespecially shown, and its explanation is omitted.

APPLICATION EXAMPLE 1

FIG. 19 is a schematic diagram showing that a sample 107 a is surroundedwith a gelled wall 106, and is moved and secured.

First, in FIG. 19, the solution including a heat sensitive substance andthe sample 107 a is flowed in from an inlet (In1) shown at the top ofFIG. 17 to the glass plate 102, and the solution is flowed out from anoutlet (Out1) shown at the bottom of FIG. 19. Then the liquid channelsare formed by stimulating the metal pieces 103 being heat sensitivesubstances to turn the heat sensitive substance into a gel (the liquidchannel in which the solution flows is not shown in FIG. 19).

When the sample 107 a flowing in this liquid channel is detected by thedetecting means 122, the metal pieces 103 are irradiated with the laserto form the gelled wall 106 caused by the heat sensitive substance so asto surround the sample 107 a. Next, as indicated by the arrow, thesample 107 a surrounded with the wall 106 is moved to an area whereheaters (not shown) are located in the glass plate 102. Thereafter thesample 107 a′ is fixed, being surrounded with the wall 106′.

The sample 107 a is moved as follows: first the metal piece just to theright of the metal piece causing the gelled wall 106 is gelled, and thenthe gelled wall 106 to its left is turned into a sol. Thus the entirewall can be moved by one metal piece. By repeating this many times, thesample 107 a can be moved to an area where heaters are located in theglass plate 102, while the sample 107 is surrounded with the wall 106 a.Then a thermal change of the sample 107 a′ can be observed by heatingthe sample 107 a′ using the heaters. Various biological samples such asa cell, organelle, nucleic acid, protein can be used as the sample.

APPLICATION EXAMPLE 2

By the same method as the application example 1, as shown in FIG. 20,the first sample 107 a′ is surrounded with the gelled wall 106′, andfixed at the area where heaters (not shown) are located.

Next, as shown in FIG. 20, the solution including a heat sensitivesubstance and the sample 107 b is flowed in from an inlet (In2) shown atthe top of FIG. 20 to the glass plate 102, and the solution is flowedout from an outlet (Out2) shown at the bottom of FIG. 20. The liquidchannels are formed by stimulating the metal pieces 103 to turn the heatsensitive substance into a gel (the liquid channel in which the solutionflows is not shown in FIG. 20). Thereafter a second sample 107 b issurrounded with the gelled wall 106 by the same method as applicationexample 1.

The second sample 107 b is moved to the place of the wall 106′ where thefirst sample 107 a′ is fixed. Then a part of the gelled walls 106 and106′ are each opened to put the first sample 107 a′ and the secondsample 107 b′ inside the same walls. Thereafter, as shown in FIG. 21,the wall size is changed so that the first sample 107 a′ may react withthe second sample 107 b′. Then, the first sample 107 a′ is contactedwith the second sample 107 b′ to chemically react them by using heatfrom a heater, electric field, or the like.

For the sample, various biological samples and agents such as a cell,organelle, nucleic acid, protein can be used to analyze theirinteraction or chemical reaction.

APPLICATION EXAMPLE 3

FIGS. 22 to 24 are schematic diagrams showing a condition where acertain amount of sample is collected and moved to an analyzing system,or the like.

The solution including the heat sensitive substance is flowed in frominlets (In1, In2) at the top of the FIG. 22 to the glass plate 102, andthe liquid channels are formed by irradiating arbitrary metal pieces 103being the stimulation sensitive members with a laser. Next the solutionmixed with the heat sensitive substance and the sample 107 c is flowedin from the inlets (In1, In2) at the top of the FIG. 22 to the glassplate 102. The solution flows through a predetermined channel, and flowsout from the outlets (Out1, Out2) at the bottom of FIG. 22. Afterconfirmation of the state that the solution is flowing through thechannels using a detecting means 122, the metal pieces 103 at the sideof the inlets and the outlets in the channel are irradiated with thelaser to turn the heat sensitive substance into a gel, after whichinflow of the solution is stopped. FIG. 23 shows this condition. Thus, acertain amount of sample 107 c can be held in the glass plate 102.

Next, as shown in FIG. 24, in order to flow a carrier solution from aninlet (In2′) shown at the left-hand side of FIG. 24 to the plate 102,and transfer the carrier solution through an outlet (Out2′) at theright-hand side of FIG. 24 to the analyzing device, the metal pieces 103are irradiated with the laser to turn the heat sensitive substance intothe gel, and thereafter the carrier solution is flowed in from the inlet(In2′). Thus, a certain amount of the sample 107 c can be transferredfrom the outlet (Out2′) to the analyzing device.

APPLICATION EXAMPLE 4

FIGS. 25 and 26 are schematic diagrams showing molecule separation byelectrocataphoresis.

In FIG. 25, instead of a pair of the inlet 104 c at the left side ofFIG. 13 and the outlet 104 d at the right side of FIG. 13, a pair ofelectrodes 109 and 109 are provided so as to sandwich the glass plate102 therebetween. The solution including the heat sensitive substance isflowed in from an inlet (In2) at the top of FIG. 25 to the glass plate102. The solution is flowed out from an outlet (Out2) at the bottom ofFIG. 25. To do this, the liquid channel is formed by irradiating themetal pieces 103 being stimulation sensitive members with the laser toturn the heat sensitive substance into the gel. Thereafter when thesolution mixed with the heat sensitive substance and a sample 107 d isflowed in from the inlet at the top of FIG. 25 to the glass plate 102,the solution flows through a predetermined channel.

After confirmation of the state that the solution is flowing through thechannel, using the detecting means 122, the metal pieces 103 around theinlet and the metal pieces 103 around the outlet are irradiated with thelaser to turn the heat sensitive substance into a gel. Thus the solutiondoes not flow on the glass plate 102. FIG. 26 shows this state.

Thereafter, by impressing an electric filed to the pair of electrodes109 in this state, molecule separation of sample 107 d can be performedby electrophoresis.

Embodiment 9

Hereafter, based on FIG. 27 and FIG. 28, an explanation will be given ofa nano-aperture film according to a ninth embodiment of the presentinvention. Numeral 201 denotes a nano-aperture film (i.e. a film with anano-aperture). The nano-aperture film 201 is composed of a thin filmthat does not transmit excitation light 203 which excites a fluorescentbiomolecule 202 labeled with fluorescent dye, the fluorescentbiomolecule 202 being the analysis object. The nano-aperture film 201 iscombined with a transparent plate 204 made from a material such as aglass and the nano-aperture film 201 has the thin film formed on theplate 204, the thin film being made from a material such as a metal(e.g. aluminum, chromium, gold, silver, or germanium), or siliconcarbide (SiC) by using a technique such as vapor deposition.

The nano-aperture film 201 is formed with a plurality of nano-apertures205, wherein the nano-apertures 205 are arranged at equal intervals ofeach interval d in an anteroposterior and horizontal direction. Thenano-apertures 205 are formed in a circle of diameter Φ. It should benoted that the nano-apertures 205 do not necessarily need to be in acircle. When the nano-apertures 205 are not in a circle, the diameter Φis set to the maximum opening width of the nano-apertures 205.

Moreover, the diameter Φ of the nano-aperture 205 is smaller than thewavelength λex of the excitation light 203. The diameter Φ is preferableas small as possible. That is, the smaller the diameter Φ is reduced thesmaller the region of an evanescent field 206 hereinafter described,which is advantageous for exciting the fluorescent biomolecule 202 atthe level of a single molecule. Therefore, it is desirable that thediameter Φ is 200 nm or less, and more preferably 20 nm or less.

Moreover, in order to detect the fluorescence which the fluorescentbiomolecule 202 emits at the level of a single molecule, the interval dbetween the nano-apertures 205 is made the same as or greater than theresolution of an objective lens 213 of an optical microscope, whichconstitutes a fluorescence detecting means 212 (hereinafter described),for detecting a fluorescence 207 of the fluorescent biomolecule 202.That is, when the detected light is not coherent, the resolution of theobjective lens 213 is defined by the formula: 0.61 λem/NA, wherein λemis the wavelength of the fluorescence 207, and NA is the numericalaperture of the objective lens 213. Therefore, the interval d betweenthe nano-apertures 205 satisfies for the formula: d>0.61 λem/NA. Forexample, when the wavelength λem of the fluorescence 207 is 500 nm, theresolution of the objective lens 213 is 0.61 λem/NA≈250 nm assuming thenumerical aperture (NA) of the objective lens 213 is set to a value of1.2. Therefore, when the objective lens 213 whose the numerical aperture(NA) is 1.2 is used, the fluorescence which the fluorescent biomolecule202 emits can be detected at the level of a single molecule by using thenano-aperture film 201 with the nano-apertures 205, wherein the intervald between the nano-apertures 205 is 250 nm or more.

Next, the operation is described. As shown in FIG. 28, when theexcitation light 203 is incident from the side of the plate 204 on whichthe nano-aperture film 201 is not combined, excitation light 203 leaksout from the nano-apertures 205, namely, an evanescent field 206 isgenerated. The size of this evanescent field 206 is comparable to thesize of the nano-apertures 205, and is capable of exciting fluorescentbiomolecules 202 residing in a region smaller than the wavelength λex ofthe excitation light 203 near the nano-apertures 205 and emittingfluorescence 207. Moreover, the plurality of nano-apertures 205 arespaced at more than the resolution of the objective lens 213 of theoptical microscope, so that it is possible to isolate the fluorescence207 of each fluorescent biomolecule 202 excited through eachnano-aperture 205, and to measure one molecule.

In addition, since the evanescent field 206 is attenuated over about 150nm of penetration length, the region of the evanescent field 206 isproportional to the area of the nano-aperture 205. Therefore, when thefluorescent biomolecule 202 is excited in the conventional way using theevanescent field due to the total reflection of the interface withoutallowing it to pass through the nano-aperture film 201, in order todetect the fluorescent biomolecule 202 at the level of a singlemolecule, the concentration of the fluorescent biomolecule 202 needs tobe set to 50 nM or less so that only one molecule exists within adiameter of 250 nm, which is the resolution of the objective lens 213.However, by using the nano-aperture film 201 of this invention, when thediameter of the nano-aperture 205 is 100 nm, the concentration of thefluorescent biomolecule 202 may be made to increase to about 300 nM.Furthermore, when the diameter of the nano-aperture 205 is 20 nm, theconcentration of the fluorescent biomolecule 202 may be made to increaseto about 8000 nM. That is, the concentration of the fluorescentbiomolecule 202 may be made to increase to 100 to 1000 times incomparison to the conventional concentration. Therefore, it is possibleto decrease exponentially the adverse effects where biomolecules areabsorbed nonspecifically to the surface of the glass, or the like.

As mentioned above, the nano-aperture film 201 in the above-mentionedembodiment is provided with nano-apertures 205, and comprised of a thinfilm which does not transmit light. Therefore, when the maximum openingwidth Φ of the nano-apertures 205 is made smaller than the wavelengthλex of the excitation light 203, and these nano-apertures 205 areirradiated with the excitation light 203, the evanescent field 206 isgenerated through these nano-apertures 205 so that the fluorescentbiomolecule 202 in a region smaller than the wavelength λex of theexcitation light 203 can be irradiated with the excitation light 203 byusing the evanescent field 206.

Moreover, since the nano-aperture film 201 being the thin film iscombined with the transparent plate 204, the manufacturing and handlingof the nano-aperture film 201 can be improved by supporting thenano-aperture film 201 on the plate 204. Moreover, since the plate 204is transparent, it does not prevent the transmission of excitation light203.

Furthermore, a plurality of nano-apertures 205 are provided and arrangedat substantially equal intervals, so that the fluorescence 207 of thefluorescent biomolecule 202 is observable in the arbitrarynano-apertures 205 of a plurality of nano-apertures 205. Thus alignmentby a fluorescence detecting means is easy. Moreover, when the interval dbetween the nano-apertures 205 is the same as the resolution of thefluorescence detecting means, or larger than the resolution of thefluorescence detecting means, the fluorescence 207 of each fluorescentbiomolecule 202 excited by each nano-aperture 205 can be separated, andthe interaction between biomolecules can be detected at the level of asingle molecule.

Furthermore, since the diameter Φ being the maximum opening width of thenano-aperture 205 is 200 nm or less, the diameter Φ of the nano-aperture205 can be made smaller than the wavelength λex of the excitation light203.

Embodiment 10

Next, a device for analyzing a biomolecular interaction according to atenth embodiment of the present invention will be explained withreference to FIG. 29 and FIG. 30. This device for analyzing abiomolecular interaction is equipment for analyzing the intensity of thefluorescence 207 which the fluorescent biomolecule 202 emits, or thediffusion coefficient of the fluorescent biomolecule 202, byfluorescence correlation spectroscopy (FCS) using a nano-aperture film201. Here the construction of the nano-aperture film 201 is similar tothat of the above-mentioned embodiment, and the same reference numeralsare used, and detailed description is omitted.

Numeral 211 denotes a laser being an excitation light generating meansfor generating an excitation light. A lamp instead of the laser 211 maybe used. This laser 211 is configured so that the nano-aperture film 201is irradiated with the excitation light 203 for the fluorescentbiomolecule 202. An aqueous solution 208 including the fluorescentbiomolecule 202 is held between a side where the plate 204 is combinedwith the nano-aperture film 201, and a cover glass 209, and theconstruction is such that the excitation light 203 irradiates from theside where the plate 204 is not combined with the nano-aperture film201.

The outside of the cover glass 209 is provided with a fluorescencedetecting means 212 for detecting the fluorescence 207 emitted from thefluorescent biomolecule 202. This fluorescence detecting means 212 isprovided with an objective lens 213 of a microscope (not shown), anoptical filter 214, a pinhole 215, and a detector 216. The objectivelens 213 is arranged so as to gather the fluorescence 207 emitted fromthe fluorescent biomolecule 202. The optical filter 214 is arranged soas to remove a background light such as dispersion light and to passonly the fluorescence 207. Moreover, the pinhole 215 is arranged so asto detect the fluorescence 207 from the single nano-aperture 205, and apore size of the pinhole 215 is approximately the resolution of theobjective lens 213× the magnification of the objective lens 213. Theresolution of the objective lens 213 is defined by the formula: 0.61λm/NA for a numerical aperture (NA) of the objective lens 213 asabove-mentioned. It is configured so that the fluorescence 207 passingthrough the pinhole 215 is detected with a high-sensitivity detector216, and then a detection signal thereof is processed with a digitalcounter, a digital correlation machine, or the like so as to analyzeaccording to the technique of the conventional FCS.

Next, an analysis method by using the above-mentioned device foranalyzing a biomolecular interaction will be explained. Firstly, theaqueous solution 208 including the fluorescent biomolecule 202 is addedbetween the nano-aperture film 201 and the cover glass 209, and mountedon a microscope. The excitation light 203 is incident from the back sideof the nano-aperture 205, and generates the evanescent field 206. Whenthe fluorescent biomolecule 202 passes through the evanescent field 206,the fluorescence 207 is emitted. The fluorescence 207 is gathered withthe objective lens 213, the background light such as dispersion light isremoved with the optical filter 214, and only the fluorescence 207 ispassed. Then, the fluorescence 207 which had passed through the opticalfilter 214 is passed through the pinhole 215, and only the fluorescence207 from a single nano-aperture 205 is detected by the detector 216.Then a detection signal thereof is processed with a digital counter, adigital correlation machine, or the like, and is analyzed according tothe technique of the conventional FCS.

As is apparent from the above, a device for analyzing a biomolecularinteraction according to the aforementioned embodiment comprises: thelaser 211 being the excitation light generating means for generating theexcitation light 203; the nano-aperture film 201 which comprises a thinfilm which does not transmit light, and in which the nano-apertures 205are formed, wherein a diameter Φ being a maximum opening width of thenano-aperture is smaller than the wavelength λex of the excitation light203; and the fluorescence detecting means 212 for detecting thefluorescence 207. When the nano-aperture film 201 with thenano-apertures 205, the diameter Φ of which is smaller than thewavelength λex of the excitation light 203, is irradiated with theexcitation light 203 from the laser 211, the evanescent field 206 isgenerated in the nano-apertures 205. Therefore, by using the evanescentfield 206, the fluorescent biomolecule 202 can be irradiated with theexcitation light 203 in an area smaller than the wavelength λex of theexcitation light 203, and the fluorescence 207 emitted from thefluorescent biomolecule 202 is able to be detected with the fluorescencedetecting means 212. Moreover, by irradiating the fluorescentbiomolecule 202 with the excitation light 203 in an area smaller thanthe wavelength λex of the excitation light 203, the concentration in thesolution 208 including the fluorescent biomolecule 202 can be increased.Furthermore, the influence of the nonspecific absorption of thefluorescent biomolecule 202 in the surface of the plate 204 being aglass surface, can be prevented. Thus detection or determination of thebiomolecular interaction can be performed reliably.

Moreover, a plurality of nano-apertures 205 are provided and arranged atequal intervals, and the interval d between the nano-apertures 205 isthe same as the resolution of the objective lens 213 of the fluorescencedetecting means 212, or larger than the resolution of the objective lens213. Therefore that the fluorescence 207 of the fluorescent biomolecule202 is observable in arbitrary nano-apertures 205 of a plurality ofnano-apertures 205, and hence alignment by a fluorescence detectingmeans is facilitated. Moreover, since the interval d between thenano-apertures 205 is the same as the resolution of the objective lens213 of the fluorescence detecting means 212, or larger than theresolution of the objective lens 213, the fluorescence 207 of eachfluorescent biomolecule 202 excited by each nano-aperture 205 can beseparated, and the interaction between biomolecules can be detected atthe level of a single molecule.

Furthermore, a method of analyzing a biomolecular interaction accordingto the foregoing embodiment comprises the steps of: generating anevanescent field 206 by the excitation light 203 from the nano-apertures205 smaller than a wavelength λex of the excitation light 203; excitinga fluorescent biomolecule 202 which passes through a certain region ofthe evanescent field 206 by Brownian motion; and detecting thefluorescence 207 of the fluorescent biomolecule 202. Hence, thefluorescent biomolecule 202 can be irradiated with the excitation light203 in an area smaller than the wavelength λex of the excitation light203, and the interaction between biomolecules can be detected at thelevel of a single molecule.

Embodiment 11

Next, a device for analyzing a biomolecular interaction according to aneleventh embodiment of the present invention will be explained withreference to FIG. 31 and FIG. 32. This device for analyzing abiomolecular interaction is equipment for detecting the biomolecularinteraction of fluorescent biomolecules 202 a and 202 b fromfluorescence 207 (207 a, 207 b) which the fluorescent biomolecules 202 aand 202 b labeled with fluorescence molecules having differentfluorescence wavelengths emit, by fluorescence cross-correlationspectroscopy (FCCS), using the nano-aperture film 201. The same portionsas those described in the above-mentioned embodiment are designated bythe same reference numerals, and their detailed description is omitted.

Numeral 211 denotes a laser being an excitation light generating meansfor generating an excitation light. A lamp instead of the laser 211 maybe used. This laser 211 is configured so that the nano-aperture film 201is irradiated with the excitation light 203 being common to two kinds offluorescent biomolecules 202 a and 202 b. An aqueous solution 208including the fluorescent biomolecules 202 a and 202 b is held between aside where the plate 204 is combined with the nano-aperture film 201,and a cover glass 209, and the construction is such that the excitationlight 203 irradiates from the side where the plate 204 is not combinedwith the nano-aperture film 201.

The outside of the cover glass 209 is provided with a fluorescencedetecting means 221 for detecting the fluorescence 207 (207 a, 207 b)emitted from the fluorescent biomolecules 202 a and 202 b. Thisfluorescence detecting means 221 is provided with an objective lens 213of a microscope (not shown), a pinhole 215, a dichroic mirror 222,optical filters 214 a, 214 b, and detectors 216 a, 216 b. The objectivelens 213 is arranged so as to gather the fluorescence 207 (207 a, 207 b)emitted from the fluorescent biomolecules 202 a and 202 b. The pinhole215 is arranged so as to detect the fluorescence 207 (207 a, 207 b)through the single nano-aperture 205 among the fluorescence 207 gatheredwith the objective lens 213. Moreover, a pore size of the pinhole 215 isapproximately the resolution of the objective lens 213× themagnification of the objective lens 213. The resolution of the objectivelens 213 is defined by the formula: 0.61 λem/NA for a numerical aperture(NA) of the objective lens 213 as above-mentioned.

The dichroic mirror 222 is used to transmit only a specific wavelengthregion and reflect other regions. The dichroic mirror 222 is arrangedhere so as to transmit the fluorescence 207 a emitted from thefluorescent biomolecule 202 a among the fluorescence 207 which passesthrough the pinhole 215, and reflect the fluorescence 207 b emitted fromthe fluorescent biomolecule 202 b. Moreover, optical filters 214 a and214 b are arranged so as to remove background light such as dispersionlight among the light containing the fluorescences 207 a and 207 btransmitted and reflected by the dichroic mirror 222, and pass onlyfluorescences 207 a and 207 b, respectively. Then, the inventionaccording to this embodiment comprises the steps of: detecting thefluorescences 207 a, 207 b which have passed through the optical filters214 a and 214 b, with the high-sensitivity detectors 216 a and 216 b,respectively; processing a detection signal thereof with a digitalcounter or a digital correlation machine, or the like; cross-correlatingfluorescences 207 a and 207 b according to the technique of theconventional FCCS; and detecting the association of the fluorescentbiomolecule 202 a and the fluorescent biomolecule 202 b.

Next, an analysis method by using the above-mentioned device foranalyzing a biomolecular interaction will be explained. Firstly, theaqueous solution 208 including the fluorescent biomolecules 202 a and202 b is added between the nano-aperture film 201 and the cover glass209, and mounted on a microscope. The excitation light 203 is incidentfrom the back side of the nano-aperture 205, and generates theevanescent field 206. When the fluorescent biomolecules 202 a and 202 bpass through the evanescent field 206, the fluorescent biomolecules 202a and 202 b are excited, and then the fluorescences 207 a and 207 b areemitted, respectively. The fluorescences 207 a and 207 b are gatheredwith the objective lens 213, and their lights are passed through thepinhole 215 so as to pass only the fluorescence 207 a and 207 b from thesingle nano-aperture 205. Then, the fluorescence 207 a and 207 b whichhad passed the pinhole 215 is separated with a dichroic mirror 222. Thatis, the fluorescence 207 a is transmitted through the dichroic mirror222, and the fluorescence 207 b is reflected by the dichroic mirror 222.In addition, as for the fluorescences 207 a and 207 b separated by thedichroic mirror 222, background light such as dispersion light isremoved by the optical filters 214 a and 214 b, respectively. Then, thefluorescences 207 a and 207 b which have passed through the opticalfilters 214 a and 214 b are detected by the detectors 216 a and 216 b,respectively.

As shown in the center nano-aperture 205 in FIG. 31, when thefluorescent biomolecule 202 a and the fluorescent biomolecule 202 b bindtogether, the fluorescences 207 a and 207 b of the fluorescentbiomolecules 202 a and 202 b are observed simultaneously. On the otherhand, as shown in the nano-apertures 205 of the opposite ends in FIG.31, when the two fluorescent biomolecules 202 a and 202 b are not boundtogether, only one fluorescence 207 (the fluorescence 207 a orfluorescence 207 b) is detected. The detection signal thereof isprocessed with a digital counter, a digital correlation machine, or thelike, and cross-correlation of the fluorescences 207 a and 207 baccording to the technique of the conventional FCCS is made so as todetect the association of the fluorescent biomolecule 202 a and thefluorescent biomolecule 202 b.

As explained above, a device for analyzing a biomolecular interactionaccording to the foregoing embodiment comprises: the laser 211 being theexcitation light generating means for generating the excitation light203; the nano-aperture film 201 which comprises a thin film which doesnot transmit light, and in which the nano-apertures 205 are formed,wherein a diameter Φ being a maximum opening width of the nano-apertureis smaller than the wavelength λex of the excitation light 203; and thefluorescence detecting means 221 for detecting the fluorescence 207 (207a, 207 b). When the nano-aperture film 201 with the nano-apertures 205,the diameter Φ of which is smaller than the wavelength λex of theexcitation light 203, is irradiated with the excitation light 203 fromthe laser 211, the evanescent field 206 is generated in thenano-apertures 205. Therefore, by using the evanescent field 206, thefluorescent biomolecules 202 a and 202 b can be irradiated with theexcitation light 203 in an area smaller than the wavelength λex of theexcitation light 203, and the fluorescences 207 a and 207 b emitted fromthe fluorescent biomolecules 202 a and 202 b is able to be detected withthe fluorescence detecting means 221. Moreover, by irradiating thefluorescent biomolecules 202 a and 202 b with the excitation light 203in an area smaller than the wavelength λex of the excitation light 203,the concentration in the solution 208 including the fluorescentbiomolecules 202 a and 202 b can be increased. Furthermore, theinfluence of the nonspecific absorption of the fluorescent biomolecules202 a and 202 b in the surface of the plate 204 being a glass surface,can to be prevented. Thus detection or determination of the biomolecularinteraction can be performed reliably.

Moreover, a plurality of nano-apertures 205 are provided and arranged atequal intervals, and the interval d between the nano-apertures 205 isthe same as the resolution of the objective lens 213 of the fluorescencedetecting means 221, or larger than the resolution of the objective lens213. Therefore the fluorescences 207 a and 207 b of the fluorescentbiomolecules 202 a and 202 b are observable in arbitrary nano-apertures205 of a plurality of nano-apertures 205, and hence alignment by afluorescence detecting means is facilitated. Moreover, since theinterval d between the nano-apertures 205 is the same as the resolutionof the objective lens 213 of the fluorescence detecting means 221 orlarger than the resolution of the objective lens 213, the fluorescences207 a and 207 b of each fluorescent biomolecules 202 a and 202 b excitedby each nano-aperture 205 can be separated, and the interaction betweenbiomolecules can be detected at the level of a single molecule.

Furthermore, a method of analyzing a biomolecular interaction accordingto the foregoing embodiment comprises the steps of: generating theevanescent field 206 by the excitation light 203 from the nano-apertures205 smaller than a wavelength λex of the excitation light 203; excitingthe fluorescent biomolecules 202 a and 202 b which pass through acertain region of the evanescent field 206 by Brownian motion; anddetecting the fluorescences 207 a and 207 b of the fluorescentbiomolecules 202 a and 202 b. Hence, the fluorescent biomolecules 202 aand 202 b can be irradiated with the excitation light 203 in an areasmaller than the wavelength λex of the excitation light 203, and theinteraction between biomolecules can be detected at the level of asingle molecule.

Embodiment 12

Next, a device for analyzing a biomolecular interaction according to atwelfth embodiment of the present invention will be explained withreference to FIG. 33 and FIG. 34. This device for analyzing abiomolecular interaction is equipment for detecting the biomolecularinteraction of fluorescent biomolecules 202 a and 202 b fromfluorescence 207 b which the fluorescent biomolecule 202 b emits, byusing the fluorescent biomolecules 202 a and 202 b labeled withfluorescence molecules having different fluorescence wavelengths, by asingle fluorescent molecule imaging method or multi fluorescent moleculemicrometry, using the nano-aperture film 201. The same portions as thosedescribed in the above-mentioned embodiment are designated by the samereference numerals, and their detailed description is omitted.

Numeral 211 denotes a laser being an excitation light generating meansfor generating an excitation light. A lamp instead of the laser 211 maybe used. This laser 211 is configured so that the nano-aperture film 201is irradiated with the excitation light 203. The fluorescent biomolecule202 a is attached to the nano-aperture 205, and an aqueous solution 208including the fluorescent biomolecule 220 b is held between a side wherethe plate 204 is combined with the nano-aperture film 201, and a coverglass 209. The construction is such that the excitation light 203irradiates from the side where the plate 204 is not combined with thenano-aperture film 201.

The outside of the cover glass 209 is provided with a fluorescencedetecting means 231 for detecting the fluorescence 207 (207 a, 207 b)emitted from the fluorescent biomolecules 202 a and 202 b. Thisfluorescence detecting means 231 is provided with an objective lens 213of a microscope (not shown), an optical filter 214, and a camera 232.The objective lens 213 is arranged so as to gather the fluorescence 207(207 a, 207 b) emitted from the fluorescent biomolecules 202 a and 202b. The optical filter 214 is arranged so as to remove background light,such as dispersion light and pass only the fluorescence 207 (207 a, 207b). The resolution of the objective lens 213 is defined by the formula:0.61 λem/NA for a numerical aperture (NA) of the objective lens 213 asabove-mentioned. In addition, the image of the fluorescence 207 (207 a,207 b) which has passed through the optical filter 214 is arranged so asto be detected by the high-sensitivity camera 232.

Next, an analysis method by using the above-mentioned device foranalyzing a biomolecular interaction will be explained. Firstly, theaqueous solution 208 including the fluorescent biomolecule 202 is addedbetween the nano-aperture film 201 and the cover glass 209, and thefluorescent biomolecule 202 is allowed to attach to the nano-aperture205. The fluorescent biomolecule 202 unattached to the nano-aperture 205is washed away, and then the aqueous solution 208 including anotherfluorescent biomolecule 202 b is added between the nano-aperture film201 and the cover glass 209, and mounted on a microscope. The excitationlight 203 is incident from the back side of the nano-aperture 205, andgenerates the evanescent field 206.

Firstly, the fluorescent biomolecule 202 a is excited, the image of thefluorescence 207 a is observed with the camera 232, and the position ofthe fluorescent biomolecule 202 a is confirmed. In the case of thesingle fluorescent molecule imaging method, the number of the attachedfluorescent biomolecules 202 a is adjusted so as to be one or less foreach nano-aperture 205. In case of multi fluorescent moleculemicrometry, it is possible to set the number of the attached fluorescentbiomolecules 202 a to any value of one or more for each nano-aperture205.

Next, by exciting another fluorescent biomolecule 202 b and capturingthe image of the fluorescence 207 b by the camera 232, the situation ofinteractions, such as association and dissociation between thefluorescent biomolecule 202 a attached to the nano-aperture 205 and theanother fluorescent biomolecule 202 b can be observed. In the case ofthe single fluorescent molecule imaging method, the analysis can beperformed for each nano-aperture 205. Moreover, by using the same deviceas the device used by the single fluorescent molecule imaging method, ifthe molecules for observing are increased, observation by multifluorescent molecule micrometry can be performed. However, in the caseof multi fluorescent molecule micrometry, the fluorescence 207 b fromtwo or more nano-apertures 205 is detected simultaneously. Therefore, inthe case of multi fluorescent molecule micrometry, detectors, such as aphotomultiplier tube, in addition to camera 232 may be used. By theabove method, an association rate constant, a dissociation rateconstant, a dissociation constant, or the like, for the biomolecularinteraction can be obtained.

As explained above, a device for analyzing a biomolecular interactionaccording to the foregoing embodiment comprises: the laser 211 being theexcitation light generating means for generating the excitation light203; the nano-aperture film 201 which comprises a thin film which doesnot transmit light, and in which the nano-apertures 205 are formed,wherein a diameter Φ being a maximum opening width of the nano-apertureis smaller than the wavelength % ex of the excitation light 203; and thefluorescence detecting means 231 for detecting the fluorescence 207 (207a, 207 b). When the nano-aperture film 201 with the nano-apertures 205,the diameter Φ of which is smaller than the wavelength λex of theexcitation light 203, is irradiated with the excitation light 203 fromthe laser 211, the evanescent field 206 is generated in thenano-apertures 205. Therefore, by using the evanescent field 206, thefluorescent biomolecules 202 a and 202 b can be irradiated with theexcitation light 203 in an area smaller than the wavelength λex of theexcitation light 203, and the fluorescences 207 a and 207 b emitted fromthe fluorescent biomolecules 202 a and 202 b are able to be detectedwith the fluorescence detecting means 231. Moreover, by irradiating thefluorescent biomolecules 202 a and 202 b with the excitation light 203in an area smaller than the wavelength λex of the excitation light 203,the concentration in the solution 208 including the fluorescentbiomolecules 202 a and 202 b can be increased. Furthermore, theinfluence of the nonspecific absorption of the fluorescent biomolecules202 a and 202 b in the surface of the plate 204 being a glass surface,can be prevented. Thus detection or determination of the biomolecularinteraction can be performed reliably.

Moreover, a plurality of nano-apertures 205 are provided and arranged atequal intervals, and the interval d between the nano-apertures 205 isthe same as the resolution of the objective lens 213 of the fluorescencedetecting means 231, or larger than the resolution of the objective lens213. Therefore the fluorescence 207 of the fluorescent biomolecules 202a and 202 b is observable in arbitrary nano-apertures 205 of a pluralityof nano-apertures 205, and hence alignment by a fluorescence detectingmeans is facilitated. Moreover, since the interval between thenano-apertures 205 is the same as the resolution of the objective lens213 of the fluorescence detecting means 231 or larger than theresolution of the objective lens 213, the fluorescences 207 a and 207 bof each fluorescent biomolecule 202 a and 202 b respectively excited byeach nano-aperture 205 can be separated, and the interaction betweenbiomolecules can be detected at the level of a single molecule.

Furthermore, a method of analyzing a biomolecular interaction accordingto the foregoing embodiment comprises the steps of: generating anevanescent field 206 by the excitation light 203 from the nano-aperture205 smaller than a wavelength λex of the excitation light 203; excitinga first fluorescent biomolecule 202 a attached to the nano-aperture 205,and a second fluorescent biomolecule 202 b which is in a certain regionof the evanescent field 206 and interacts with the first fluorescentbiomolecule 202 a; and detecting the fluorescences 207 a and 207 b ofthese first and second fluorescent biomolecules 202 a and 202 b,respectively. Hence, the fluorescent biomolecules 202 a and 202 b can beirradiated with the excitation light 203 in an area smaller than thewavelength λex of the excitation light 203, and the interaction betweenbiomolecules can be detected and determined at the level of a singlemolecule.

As explained in detail above, according to the foregoing embodiment, byskillfully combining known methods, such as generation of an evanescentfield from at least one nano-aperture, single fluorescent moleculeimaging method, FCS and FCCS, the invention enables solving of theconventional theoretical problems, so that it is possible to add amolecule having a concentration as high as 100 to 1000 times theconventional critical concentration into an aqueous solution. It is alsopossible to limit the influence of the nonspecific adsorption to a glassside or the like, to about 1/100 lower than before.

Moreover, the invention can determine biomolecular interaction at a highsensitivity at the level of a single molecule, and is applicable to awide range of fields such as biology, medicine, and pharmacy. Inparticular, research of the interaction between proteins is important aspost-genome research. However, according to the invention, it is alsopossible to detect biomolecular interaction at high-sensitivity,especially the interaction between proteins at the level of a singlemolecule and carry out performance analysis. Furthermore, according tothe invention, it is possible to detect a weak interaction in which thebinding constant is smaller than 10⁶ M which has previously beenimpossible. This invention is immediately applicable to DNA chips orprotein chips, and should demonstrate a significant influence in theanalysis of the interaction between proteins.

The present invention is not limited to the above-mentioned embodiments,and various modification and variations are possible within the scope ofthe present invention. Although the film with a plurality ofnano-apertures as explained here is shown, for example, the film may bea film with one nano-aperture.

1. A micro-system comprising a stimulation applying means for applyingstimulation to a liquid flowing in a liquid channel formed in a plate,the liquid flow being controlled by the stimulation from the stimulationapplying means, wherein the stimulation applying means comprises acontrol means for electrically controlling an amount of stimulationapplied to the liquid.
 2. The micro-system according to claim 1, furthercomprising a stimulation detecting means for detecting the amount ofstimulation, wherein said stimulation applying means is a heat source ora light source, and said stimulation applying means is controlled bysaid control means based on a signal from said stimulation detectingmeans.
 3. The micro-system according to claim 2, wherein said heatsource is a micro-heater.
 4. The micro-system according to claim 2,wherein said stimulation detecting means is a heat sensor provided onsaid liquid channel.
 5. The micro-system according to claim 4, whereinsaid heat sensor is a thermocouple.
 6. The micro-system according toclaim 4, wherein said heat sensor is a heat sensitive semiconductor oran infrared ray sensitive sensor.
 7. The micro-system according to claim2, wherein said light source is at least one light emitting elementinstalled in said plate.
 8. The micro-system according to claim 7,wherein said light emitting element is embedded in said plate.
 9. Themicro-system according to claim 7, wherein said light emitting elementis arranged outside said plate.
 10. The micro-system according to claim9, further comprising an optical guiding path for guiding a light fromsaid light emitting element, said optical guiding path being formedhorizontally with a surface of said plate in which said liquid channelis formed.
 11. The micro-system according to claim 7, further comprisinga plurality of light emitting elements.
 12. The micro-system accordingto claim 1, further comprising: an energy imparting means for impartingenergy to said liquid; and a change detecting means for detecting achange in a substance which causes a change by energy from said energyimparting means, wherein said stimulation applying means is controlledby said control means based on a signal from said change detectingmeans.
 13. The micro-system according to claim 12, further comprising anenergy guiding path for guiding the energy from said energy impartingmeans, said energy guiding path being formed horizontally with a surfaceof said plate.
 14. The micro-system according to claim 12, wherein saidchange detecting means is a fluorescence detecting element or a lightreceiving element.
 15. The micro-system according to claim 14, whereinsaid fluorescence detecting element or said light receiving element isarranged horizontally with the surface of said plate.
 16. Themicro-system according to claim 14, wherein said fluorescence detectingelement or said light receiving element is arranged above said liquidchannel.
 17. The micro-system according to claim 1, further comprising:a stand for mounting said plate; and a positioning means for deciding aposition of said plate on said stand.
 18. A matrix type variable liquidchannel, comprising two or more stimulation sensitive members arrangedon a plate in a pattern of a two dimensional matrix.
 19. The matrix typevariable liquid channel according to claim 18, wherein said stimulationsensitive members on said plate are arranged at certain intervals. 20.The matrix type variable liquid channel according to claim 19, wherein asize of each stimulation sensitive member ranges from 2 μm or more to 20μm or less.
 21. The matrix type variable liquid channel according toclaim 19, wherein said stimulation sensitive members are arranged atintervals from 2 μm or more to 20 μm or less.
 22. The matrix typevariable liquid channel according to claim 19, wherein said stimulationsensitive members are formed by vapor deposition, sputtering, ChemicalVapor Deposition (CVD), plating, plasma polymerization, orscreen-printing.
 23. The matrix type variable liquid channel accordingto claim 18, wherein said stimulation sensitive member is stimulated byapplying a voltage or irradiating a light thereto.
 24. A matrix typevariable liquid channel system comprising: a matrix type variable liquidchannel which comprises two or more stimulation sensitive membersarranged on a plate in a pattern of a two dimensional matrix; adetecting means for detecting a substance on said plate; a stimulationapplying means for applying stimulation to said stimulation sensitivemembers; and a control means for controlling the stimulation applyingmeans based on the signal from said detecting means.
 25. The matrix typevariable liquid channel system according to claim 24, wherein saidstimulation sensitive members on said plate are arranged at certainintervals.
 26. The matrix type variable liquid channel system accordingto claim 25, wherein a size of each stimulation sensitive member rangesfrom 2 μm or more to 20 μm or less.
 27. The matrix type variable liquidchannel system according to claim 25, wherein said stimulation sensitivemembers are arranged at intervals from 2 μm or more to 20 μm or less.28. The matrix type variable liquid channel system according to claim25, wherein said stimulation sensitive members are formed by vapordeposition, sputtering, Chemical Vapor Deposition (CVD), plating, plasmapolymerization, or screen-printing.
 29. The matrix type variable liquidchannel system according to claim 24, wherein said stimulation sensitivemember is stimulated by said stimulation applying means applyingstimulation thereto, said stimulation being application of voltage orirradiation of light.
 30. A nano-aperture film, comprising a thin filmwhich does not transmit light and in which at least one nano-aperture isformed.
 31. The nano-aperture film according to claim 30, wherein saidthin film is combined with a transparent plate.
 32. The nano-aperturefilm according to claim 30, wherein a plurality of nano-apertures areprovided and arranged at substantially equal intervals.
 33. Thenano-aperture film according to claim 30, wherein a maximum openingwidth of said nano-aperture is 200 nm or less.
 34. A device foranalyzing a biomolecular interaction comprising: an excitation lightgenerating means for generating excitation light; a nano-aperture filmwhich comprises a thin film which does not transmit light and in whichat least one nano-aperture is formed, wherein a maximum opening width ofsaid nano-aperture is smaller than the wavelength of said excitationlight; and a fluorescence detecting means for detecting fluorescence.35. The device for analyzing a biomolecular interaction according toclaim 34, wherein a plurality of nano-apertures are provided andarranged at equal intervals, and the interval between saidnano-apertures is the same as the resolution of said fluorescencedetecting means or larger than the resolution of said fluorescencedetecting means.
 36. A method of analyzing a biomolecular interaction,the method comprising the steps of: generating an evanescent field by anexcitation light from a nano-aperture smaller than a wavelength of theexcitation light; exciting a fluorescent biomolecule which passesthrough a certain region of the evanescent field by Brownian motion; anddetecting fluorescence of the fluorescent biomolecule.
 37. A method ofanalyzing a biomolecular interaction, the method comprising the stepsof: generating an evanescent field by an excitation light from anano-aperture smaller than a wavelength of the excitation light;exciting a first fluorescent biomolecule allowed to attach to thenano-aperture, and a second fluorescent biomolecule which is in acertain region of the evanescent field and interacts to said firstfluorescent biomolecule; and detecting fluorescence of these first andsecond fluorescent biomolecules, respectively.